Sodium channel blockers

ABSTRACT

The present invention relates to sodium channel blockers. The present invention also includes a variety of methods of treatment using these inventive sodium channel blockers.

CONTINUING APPLICATION INFORMATION

This application is a continuation-in-part of U.S. application Ser. No.10/076,571, filed on Feb. 19, 2002, and incorporated herein by referencein its entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to sodium channel blockers. The presentinvention also includes a variety of methods of treatment using theseinventive sodium channel blockers.

2. Description of the Background

The mucosal surfaces at the interface between the environment and thebody have evolved a number of “innate defense”, i.e., protectivemechanisms. A principal form of such innate defense is to cleanse thesesurfaces with liquid. Typically, the quantity of the liquid layer on amucosal surface reflects the balance between epithelial liquidsecretion, often reflecting anion (Cl⁻ and/or HCO₃ ⁻) secretion coupledwith water (and a cation counter-ion), and epithelial liquid absorption,often reflecting Na⁺ absorption, coupled with water and counter anion(Cl⁻ and/or HCO₃ ⁻). Many diseases of mucosal surfaces are caused by toolittle protective liquid on those mucosal surfaces created by animbalance between secretion (too little) and absorption (relatively toomuch). The defective salt transport processes that characterize thesemucosal dysfunctions reside in the epithelial layer of the mucosalsurface.

One approach to replenish the protective liquid layer on mucosalsurfaces is to “re-balance” the system by blocking Na⁺ channel andliquid absorption. The epithelial protein that mediates therate-limiting step of Na⁺ and liquid absorption is the epithelial Na⁺channel (ENaC). ENaC is positioned on the apical surface of theepithelium, i.e. the mucosal surface-environmental interface. Therefore,to inhibit ENaC mediated Na⁺ and liquid absorption, an ENaC blocker ofthe amiloride class (which blocks from the extracellular domain of ENaC)must be delivered to the mucosal surface and, importantly, be maintainedat this site, to achieve therapeutic utility. The present inventiondescribes diseases characterized by too little liquid on mucosalsurfaces and “topical” sodium channel blockers designed to exhibit theincreased potency, reduced mucosal absorption, and slow dissociation(“unbinding” or detachment) from ENaC required for therapy of thesediseases.

Chronic bronchitis (CB), including the most common lethal genetic formof chronic bronchitis, cystic fibrosis (CF), are diseases that reflectthe body's failure to clear mucus normally from the lungs, whichultimately produces chronic airways infection. In the normal lung, theprimary defense against chronic intrapulmonary airways infection(chronic bronchitis) is mediated by the continuous clearance of mucusfrom bronchial airway surfaces. This function in health effectivelyremoves from the lung potentially noxious toxins and pathogens. Recentdata indicate that the initiating problem, i.e., the “basic defect,” inboth CB and CF is the failure to clear mucus from airway surfaces. Thefailure to clear mucus reflects an imbalance between the amount ofliquid and mucin on airway surfaces. This “airway surface liquid” (ASL)is primarily composed of salt and water in proportions similar to plasma(i.e., isotonic). Mucin macromolecules organize into a well defined“mucus layer” which normally traps inhaled bacteria and is transportedout of the lung via the actions of cilia which beat in a watery, lowviscosity solution termed the “periciliary liquid” (PCL). In the diseasestate, there is an imbalance in the quantities of mucus as ASL on airwaysurfaces. This results in a relative reduction in ASL which leads tomucus concentration, reduction in the lubricant activity of the PCL, anda failure to clear mucus via ciliary activity to the mouth. Thereduction in mechanical clearance of mucus from the lung leads tochronic bacterial colonization of mucus adherent to airway surfaces. Itis the chronic retention of bacteria, the failure of local antimicrobialsubstances to kill mucus-entrapped bacteria on a chronic basis, and theconsequent chronic inflammatory responses of the body to this type ofsurface infection, that lead to the syndromes of CB and CF.

The current afflicted population in the U.S. is 12,000,000 patients withthe acquired (primarily from cigarette smoke exposure) form of chronicbronchitis and approximately 30,000 patients with the genetic form,cystic fibrosis. Approximately equal numbers of both populations arepresent in Europe. In Asia, there is little CF but the incidence of CBis high and, like the rest of the world, is increasing.

There is currently a large, unmet medical need for products thatspecifically treat CB and CF at the level of the basic defect that causethese diseases. The current therapies for chronic bronchitis and cysticfibrosis focus on treating the symptoms and/or the late effects of thesediseases. Thus, for chronic bronchitis, O-agonists, inhaled steroids,anti-cholinergic agents, and oral theophyllines and phosphodiesteraseinhibitors are all in development. However, none of these drugs treateffectively the fundamental problem of the failure to clear mucus fromthe lung. Similarly, in cystic fibrosis, the same spectrum ofpharmacologic agents is used. These strategies have been complemented bymore recent strategies designed to clear the CF lung of the DNA(“Pulmozyme”; Genentech) that has been deposited in the lung byneutrophils that have futilely attempted to kill the bacteria that growin adherent mucus masses and through the use of inhaled antibiotics(“TOBI”) designed to augment the lungs' own killing mechanisms to ridthe adherent mucus plaques of bacteria. A general principle of the bodyis that if the initiating lesion is not treated, in this case mucusretention/obstruction, bacterial infections became chronic andincreasingly refractory to antimicrobial therapy. Thus, a major unmettherapeutic need for both CB and CF lung diseases is an effective meansof re-hydrating airway mucus (i.e., restoring/expanding the volume ofthe ASL) and promoting its clearance, with bacteria, from the lung.

R. C. Boucher, in U.S. Pat. No. 6,264,975, describes the use ofpyrazinoylguanidine sodium channel blockers for hydrating mucosalsurfaces. These compounds, typified by the well-known diureticsamiloride, benzamil, and phenamil, are effective. However, thesecompounds suffer from the significant disadvantage that they are (1)relatively impotent, which is important because the mass of drug thatcan be inhaled by the lung is limited; (2) rapidly absorbed, whichlimits the half-life of the drug on the mucosal surface; and (3) arefreely dissociable from ENaC. The sum of these disadvantages embodied inthese well known diuretics produces compounds with insufficient potencyand/or effective half-life on mucosal surfaces to have therapeuticbenefit for hydrating mucosal surfaces.

Clearly, what is needed are drugs that are more effective at restoringthe clearance of mucus from the lungs of patients with CB/CF. The valueof these new therapies will be reflected in improvements in the qualityand duration of life for both the CF and the CB populations.

Other mucosal surfaces in and on the body exhibit subtle differences inthe normal physiology of the protective surface liquids on theirsurfaces but the pathophysiology of disease reflects a common theme,i.e., too little protective surface liquid. For example, in xerostomia(dry mouth) the oral cavity is depleted of liquid due to a failure ofthe parotid sublingual and submandibular glands to secrete liquiddespite continued Na⁺ (ENaC) transport mediated liquid absorption fromthe oral cavity. Similarly, keratoconjunctivitis sira (dry eye) iscaused by failure of lacrimal glands to secrete liquid in the face ofcontinued Na⁺ dependent liquid absorption on conjunctional surfaces. Inrhinosinusitis, there is an imbalance, as in CB, between mucin secretionand relative ASL depletion. Finally, in the gastrointestinal tract,failure to secrete Cl— (and liquid) in the proximal small intestine,combined with increased Na⁺ (and liquid) absorption in the terminalileum leads to the distal intestinal obstruction syndrome (DIOS). Inolder patients excessive Na⁺ (and volume) absorption in the descendingcolon produces constipation and diverticulitis.

Fifty million Americans and hundreds of millions of others around theworld suffer from high blood pressure and the subsequent sequale leadingto congestive heart failure and increasing mortality. It is the WesternWorld's leading killer and there is a need there for new medicines totreat these diseases. Thus, in addition, some of the novel sodiumchannel blockers of this invention can be designed to target the kidneyand as such they may be used as diuretics for the treatment ofhypertension, congestive heart failure (CHF) and other cardiovasculardiseases. These new agents may be used alone or in combination withbeta-blockers, ACE inhibitors, HMGCoA reductase inhibitors, calciumchannel blockers and other cardiovascular agents.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide compounds that aremore potent and/or absorbed less rapidly from mucosal surfaces, and/orare less reversible as compared to known compounds.

It is another aspect of the present invention to provide compounds thatare more potent and/or absorbed less rapidly and/or exhibit lessreversibility, as compared to compounds such as amilorde, benzamil, andphenamil. Therefore, the compounds will give a prolonged pharmacodynamichalf-life on mucosal surfaces as compared to known compounds.

It is another object of the present invention to provide compounds whichare (1) absorbed less rapidly from mucosal surfaces, especially airwaysurfaces, as compared to known compounds and; (2) when absorbed frommusosal surfaces after administration to the mucosal surfaces, areconverted in vivo into metabolic derivatives thereof which have reducedefficacy in blocking sodium channels as compared to the administeredparent compound. It is another object of the present invention toprovide compounds that are more potent and/or absorbed less rapidlyand/or exhibit less reversibility, as compared to compounds such asamiloride, benzamil, and phenamil. Therefore, such compounds will give aprolonged pharmacodynamic half-life on mucosal surfaces as compared toprevious compounds.

It is another object of the present invention to provide compounds thattarget the kidney for use in the treatment of cardiovascular disease.

It is another object of the present invention to provide methods oftreatment that take advantage of the pharmacological properties of thecompounds described above.

In particular, it is an object of the present invention to providemethods of treatment which rely on rehydration of mucosal surfaces.

In particular, it is an object of the present invention to providemethods of treating cardiovascular disease.

The objects of the present invention may be accomplished with a class ofpyrazinoylguanidine compounds represented by formula (I):

where

X is hydrogen, halogen, trifluoromethyl, lower alkyl, unsubstituted orsubstituted phenyl, lower alkyl-thio, phenyl-lower alkyl-thio, loweralkyl-sulfonyl, or phenyl-lower alkyl-sulfonyl;

Y is hydrogen, hydroxyl, mercapto, lower alkoxy, lower alkyl-thio,halogen, lower alkyl, unsubstituted or substituted mononuclear aryl, or—N(R²)₂;

R¹ is hydrogen or lower alkyl;

each R² is, independently, —R⁷, —(CH₂)_(m)—OR⁸, —(CH₂)_(m)—NR⁷R¹⁰,—(CH₂)_(n)(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸, —(CH₂CH₂O)_(m)—R⁸,—(CH₂CH₂O)_(m)—CH₂CH₂NR⁷R¹⁰, —(CH₂)_(n)—C(═O)NR⁷R¹⁰,—(CH₂)_(n)-Z_(g)—R⁷, —(CH₂)_(m)—NR¹⁰—CH₂(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸,—(CH₂)_(n)—CO₂R⁷, or

R³ and R⁴ are each, independently, hydrogen, a group represented byformula (A), lower alkyl, hydroxy lower alkyl, phenyl, phenyl-loweralkyl, (halophenyl)-lower alkyl, lower-(alkylphenylalkyl), lower(alkoxyphenyl)-lower alkyl, naphthyl-lower alkyl, or pyridyl-loweralkyl, with the proviso that at least one of R³ and R⁴ is a grouprepresented by formula (A):

where

each R^(L) is, independently, —R⁷, —(CH₂)_(n)—OR⁸, —O—(CH₂)_(m)—OR⁸,—(CH₂)_(n)—NR⁷R¹⁰, —O—(CH₂)_(m)—NR⁷R¹⁰,—(CH₂)_(n)(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸,—O—(CH₂)_(m)(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸, —(CH₂CH₂O)_(m)—R⁸,—O—(CH₂CH₂O)_(m)—R⁸, —(CH₂CH₂O)_(m)—CH₂CH₂NR⁷R¹⁰,—O—(CH₂CH₂O)_(m)—CH₂CH₂NR⁷R¹⁰, —(CH₂)_(n)—C(═O)NR⁷R¹⁰,—O—(CH₂)_(m)—C(═O)NR⁷R¹⁰, —(CH₂)_(n)-(Z)_(g)—R⁷,—O—(CH₂)_(m)-(Z)_(g)—R⁷, —(CH₂)_(n)—NR¹⁰—CH₂(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸,—O—(CH₂)_(m)—NR¹⁰—CH₂(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸, —(CH₂)_(n)—CO₂R⁷,—O—(CH₂)_(m)—CO₂R⁷, —OSO₃H, —O-glucuronide, —O-glucose,

each o is, independently, an integer from 0 to 10;

each p is an integer from 0 to 10;

with the proviso that the sum of o and p in each contiguous chain isfrom 1 to 10;

each x is, independently, O, NR¹⁰, C(═O), CHOH, C(═N—R¹⁰),

CHNR⁷R¹⁰, or represents a single bond;

each R⁵ is, independently, —(CH₂)_(m)—OR⁸, —O—(CH₂)_(m)—OR⁸,—(CH₂)_(n)—NR⁷R¹⁰, —O—(CH₂)_(m)—NR⁷R¹⁰,—(CH₂)_(n)(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸,—O—(CH₂)_(m)(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸, —(CH₂CH₂O)_(m)—R⁸,—O—(CH₂CH₂O)_(m)—R⁸, —(CH₂CH₂O)_(m)—CH₂CH₂NR⁷R¹⁰,—O—(CH₂CH₂O)_(m)—CH₂CH₂NR⁷R¹⁰, —(CH₂)_(n)—C(O)NR⁷R¹⁰,—O—(CH₂)_(m)—C(═O)NR⁷R¹⁰, —(CH₂)_(n)-(Z)_(g)—R⁷,—O—(CH₂)_(m)-(Z)_(g)—R⁷, —(CH₂)_(n)—NR¹⁰—CH₂(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸,—O—(CH₂)_(m)—NR¹⁰—CH₂(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸, —(CH₂)_(n)—CO₂R⁷,—O—(CH₂)_(m)—CO₂R⁷, —OSO₃H, —O-glucuronide, —O-glucose,

each R⁶ is, independently, —R⁷, —OR¹¹, —N(R⁷)₂, —(CH₂)_(m)—OR⁸,—O—(CH₂)_(m)—OR⁸, —(CH₂)_(n)—NR⁷R¹⁰, —O—(CH₂)_(m)—NR⁷R¹⁰,—(CH₂)_(n)(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸,—O—(CH₂)_(m)(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸, —(CH₂CH₂O)_(m)—R⁸,—O—(CH₂CH₂O)_(m)—R⁸, —(CH₂CH₂O)_(m)—CH₂CH₂NR⁷R¹⁰,—O—(CH₂CH₂O)_(m)—CH₂CH₂NR⁷R¹⁰, —(CH₂)_(n)—C(═O)NR⁷R¹⁰,—O—(CH₂)_(m)—C(═O)NR⁷R¹⁰, —(CH₂)_(n)-(Z)_(g)—R⁷,—O—(CH₂)_(m)-(Z)_(g)—R⁷, —(CH₂)_(n)—NR¹⁰—CH₂(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸,—O—(CH₂)_(m)—NR¹⁰—CH₂(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸, —(CH₂)_(n)—CO₂R⁷,—O—(CH₂)_(m)—CO₂R⁷, —OSO₃H, —O-glucuronide, —O-glucose,

where when two R⁶ are —OR¹¹ and are located adjacent to each other on aphenyl ring, the alkyl moieties of the two R⁶ may be bonded together toform a methylenedioxy group;

-   -   each R⁷ is, independently, hydrogen or lower alkyl;    -   each R⁸ is, independently, hydrogen, lower alkyl, —C(═O)—R¹¹,        glucuronide, 2-tetrahydropyranyl, or

-   -   each R⁹ is, independently, —CO₂R⁷, —CON(R⁷)₂, —SO₂CH₃, or        —C(═O)R⁷;    -   each R¹⁰ is, independently, —H, —SO₂CH₃, —CO₂R⁷, —C(═O)NR⁷R⁹,        —C(═O)R⁷, or —CH₂—(CHOH)_(n)—CH₂OH;    -   each Z is, independently, CHOH, C(═O), CHNR⁷R¹⁰, C═NR¹⁰, or        NR¹⁰;    -   each R¹¹ is, independently, lower alkyl;    -   each g is, independently, an integer from 1 to 6;    -   each m is, independently, an integer from 1 to 7;    -   each n is, independently, an integer from 0 to 7;    -   each Q is, independently, C—R⁵, C—R⁶, or a nitrogen atom,        wherein at    -   most three Q in a ring are nitrogen atoms;

or a pharmaceutically acceptable salt thereof, and

inclusive of all enantiomers, diastereomers, and racemic mixturesthereof.

The present also provides pharmaceutical compositions which contain acompound described above.

The present invention also provides a method of promoting hydration ofmucosal surfaces, comprising:

administering an effective amount of a compound represented by formula(I) to a mucosal surface of a subject.

The present invention also provides a method of restoring mucosaldefense, comprising:

topically administering an effective amount of compound represented byformula (I) to a mucosal surface of a subject in need thereof.

The present invention also provides a method of blocking ENaC,comprising:

contacting sodium channels with an effective amount of a compoundrepresented by formula (I).

The present invention also provides a method of promoting mucusclearance in mucosal surfaces, comprising:

administering an effective amount of a compound represented by formula(I) to a mucosal surface of a subject.

The present invention also provides a method of treating chronicbronchitis, comprising:

administering an effective amount of a compound represented by formula(I) to a subject in need thereof.

The present invention also provides a method of treating cysticfibrosis, comprising:

administering an effective amount of compound represented by formula (I)to a subject in need thereof.

The present invention also provides a method of treating rhinosinusitis,comprising:

administering an effective amount of a compound represented by a formula(I) to a subject in need thereof.

The present invention also provides a method of treating nasaldehydration, comprising:

administering an effective amount of a compound represented by formula(I) to the nasal passages of a subject in need thereof.

In a specific embodiment, the nasal dehydration is brought on byadministering dry oxygen to the subject.

The present invention also provides a method of treating sinusitis,comprising:

administering an effective amount of a compound represented by formula(I) to a subject in need thereof.

The present invention also provides a method of treating pneumonia,comprising:

administering an effective amount of a compound represented by formula(I) to a subject in need thereof.

The present invention also provides a method of preventingventilator-induced pneumonia, comprising:

administering an effective compound represented by formula (I) to asubject by means of a ventilator.

The present invention also provides a method of treating asthma,comprising:

administering an effective amount of a compound represented by formula(I) to a subject in need thereof.

The present invention also provides a method of treating primary ciliarydyskinesia, comprising:

administering an effective amount of a compound represented by formula(I) to a subject in need thereof.

The present invention also provides a method of treating otitis media,comprising:

administering an effective amount of a compound represented by formula(I) to a subject in need thereof.

The present invention also provides a method of inducing sputum fordiagnostic purposes, comprising:

administering an effective amount of compound represented by formula (I)to a subject in need thereof.

The present invention also provides a method of treating chronicobstructive pulmonary disease, comprising:

administering an effective amount of a compound represented by formula(I) to a subject in need thereof.

The present invention also provides a method of treating emphysema,comprising:

administering an effective amount of a compound represented by formula(I) to a subject in need thereof.

The present invention also provides a method of treating dry eye,comprising:

administering an effective amount of a compound represented by formula(I) to the eye of the subject in need thereof.

The present invention also provides a method of promoting ocularhydration, comprising:

administering an effective amount of a compound represented by formula(I) to the eye of the subject.

The present invention also provides a method of promoting cornealhydration, comprising:

administering an effective amount of a compound represented by formula(I) to the eye of the subject.

The present invention also provides a method of treating Sjögren'sdisease, comprising:

administering an effective amount of compound represented by formula (I)to a subject in need thereof.

The present invention also provides a method of treating vaginaldryness, comprising:

administering an effective amount of a compound represented by formula(I) to the vaginal tract of a subject in need thereof.

The present invention also provides a method of treating dry skin,comprising:

administering an effective amount of a compound represented by formula(I) to the skin of a subject in need thereof.

The present invention also provides a method of treating dry mouth(xerostomia), comprising:

administering an effective amount of compound represented by formula (I)to the mouth of the subject in need thereof.

The present invention also provides a method of treating distalintestinal obstruction syndrome, comprising:

administering an effective amount of compound represented by formula (I)to a subject in need thereof.

The present invention also provides a method of treating esophagitis,comprising:

administering an effective amount of a compound represented by formula(I) to a subject in need thereof.

The present invention also provides a method of treating constipation,comprising:

administering an effective amount of a compound represented by formula(I) to a subject in need thereof. In one embodiment of this method, thecompound is administered either orally or via a suppository or enema.

The present invention also provides a method of treating chronicdiverticulitis comprising:

administering an effective amount of a compound represented by formula(I) to a subject in need thereof.

The present invention also provides a method of treating hypertension,comprising administering the compound represented by formula (I) to asubject in need thereof.

The present invention also provides a method of reducing blood pressure,comprising administering the compound represented by formula (I) to asubject in need thereof.

The present invention also provides a method of treating edema,comprising administering the compound represented by formula (I) to asubject in need thereof.

The present invention also provides a method of promoting diuresis,comprising administering the compound represented by formula (I) to asubject in need thereof.

The present invention also provides a method of promoting natriuresis,comprising administering the compound represented by formula (I) to asubject in need thereof.

The present invention also provides a method of promoting saluresis,comprising administering the compound represented by formula (I) to asubject in need thereof.

BRIEF DESCRIPTION OF THE FIGURES

A more complete appreciation of the invention and many of the attendantadvantages thereof will be readily obtained as the same becomes betterunderstood by reference to the following detailed description consideredin conjunction with the following figures:

FIG. 1: Effect of a compound of the present invention on MCC at t=0 hrsas described in Example 32 herein.

FIG. 2: Effect of a compound of the present invention on MCC at t=4 hrsas described in Example 32 herein.

FIG. 3: Effect of a compound of the present invention on MCC at t=0 hrsas described in Example 32 herein.

FIG. 4: Effect of a compound of the present invention on MCC at t=4 hrsas described in Example 32 herein.

FIG. 5: Effect of a compound of the present invention on MCC at t=0 hrsas described in Example 32 herein.

FIG. 6: Effect of a compound of the present invention on MCC at t=4 hrsas described in Example 32 herein.

FIG. 7: Effect of a compound of the present invention on MCC at t=0 hrsas described in Example 32 herein.

FIG. 8: Effect of a compound of the present invention on MCC at t=4 hrsas described in Example 32 herein.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based on the discovery that the compounds offormula (I) are more potent and/or, absorbed less rapidly from mucosalsurfaces, especially airway surfaces, and/or less reversible frominteractions with ENaC as compared to compounds such as amiloride,benzamil, and phenamil. Therefore, the compounds of formula (I) have alonger half-life on mucosal surfaces as compared to these compounds.

The present invention is also based on the discovery that certaincompounds embraced by formula (I) are converted in vivo into metabolicderivatives thereof that have reduced efficacy in blocking sodiumchannels as compared to the parent administered compound, after they areabsorbed from mucosal surfaces after administration. This importantproperty means that the compounds will have a lower tendency to causeundesired side-effects by blocking sodium channels located at untargetedlocations in the body of the recipient, e.g., in the kidneys.

The present invention is also based on the discovery that certaincompounds embraced by formula (I) target the kidney and thus may be usedas cardiovascular agents.

In the compounds represented by formula (I), X may be hydrogen, halogen,trifluoromethyl, lower alkyl, lower cycloalkyl, unsubstituted orsubstituted phenyl, lower alkyl-thio, phenyl-lower alkyl-thio, loweralkyl-sulfonyl, or phenyl-lower alkyl-sulfonyl. Halogen is preferred.

Examples of halogen include fluorine, chlorine, bromine, and iodine.Chlorine and bromine are the preferred halogens. Chlorine isparticularly preferred. This description is applicable to the term“halogen” as used throughout the present disclosure.

As used herein, the term “lower alkyl” means an alkyl group having lessthan 8 carbon atoms. This range includes all specific values of carbonatoms and subranges there between, such as 1, 2, 3, 4, 5, 6, and 7carbon atoms. The term “alkyl” embraces all types of such groups, e.g.,linear, branched, and cyclic alkyl groups. This description isapplicable to the term “lower alkyl” as used throughout the presentdisclosure. Examples of suitable lower alkyl groups include methyl,ethyl, propyl, cyclopropyl, butyl, isobutyl, etc.

Substituents for the phenyl group include halogens. Particularlypreferred halogen substituents are chlorine and bromine.

Y may be hydrogen, hydroxyl, mercapto, lower alkoxy, lower alkyl-thio,halogen, lower alkyl, lower cycloalkyl, mononuclear aryl, or —N(R²)₂.The alkyl moiety of the lower alkoxy groups is the same as describedabove. Examples of mononuclear aryl include phenyl groups. The phenylgroup may be unsubstituted or substituted as described above. Thepreferred identity of Y is —N(R²)₂. Particularly preferred are suchcompounds where each R² is hydrogen.

R¹ may be hydrogen or lower alkyl. Hydrogen is preferred for R¹.

Each R² may be, independently, —R⁷, —(CH₂)_(m)—OR⁸, —(CH₂)_(m)—NR⁷R¹⁰,—(CH₂)_(n)(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸, —(CH₂CH₂O)_(m)—R⁸,—(CH₂CH₂O)_(m)—CH₂CH₂NR⁷R¹⁰, —(CH₂)_(n)—C(═O)NR⁷R¹⁰—(CH₂)_(n)-Z_(g)—R⁷,—(CH₂)_(m)—NR¹⁰—CH₂(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸, —(CH₂)_(n)—CO₂R⁷, or

Hydrogen and lower alkyl, particularly C₁-C₃ alkyl are preferred for R².Hydrogen is particularly preferred.

R³ and R⁴ may be, independently, hydrogen, a group represented byformula (A), lower alkyl, hydroxy lower alkyl, phenyl, phenyl-loweralkyl, (halophenyl)-lower alkyl, lower-(alkylphenylalkyl), lower(alkoxyphenyl)-lower alkyl, naphthyl-lower alkyl, or pyridyl-loweralkyl, provided that at least one of R³ and R⁴ is a group represented byformula (A).

Preferred compounds are those where one of R³ and R⁴ is hydrogen and theother is represented by formula (A).

In formula (A), the moiety —(C(R^(L))₂)_(o)-x-(C(R^(L))₂)_(p)— definesan alkylene group bonded to the aromatic ring. The variables o and p mayeach be an integer from 0 to 10, subject to the proviso that the sum ofo and p in the chain is from 1 to 10. Thus, o and p may each be 0, 1, 2,3, 4, 5, 6, 7, 8, 9, or 10. Preferably, the sum of o and p is from 2 to6. In a particularly preferred embodiment, the sum of o and p is 4.

The linking group in the alkylene chain, x, may be, independently, O,NR¹⁰, C(═O), CHOH, C(═N—R¹⁰), CHNR⁷R¹⁰, or represents a single bond;

Therefore, when x represents a single bond, the alkylene chain bonded tothe ring is represented by the formula —(C(R^(L))₂)_(o+p)—, in which thesum o+p is from 1 to 10.

Each R^(L) may be, independently, —R⁷, —(CH₂)_(n)—OR⁸, —O—(CH₂)_(m)—OR⁸,—(CH₂)_(n)—NR⁷R¹⁰, —O—(CH₂)_(m)—NR⁷R¹⁰,—(CH₂)_(n)(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸,—O—(CH₂)_(m)(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸, —(CH₂CH₂O)_(m)—R⁸,—O—(CH₂CH₂O)_(m)—R⁸, —(CH₂CH₂O)_(m)—CH₂CH₂NR⁷R¹⁰,—O—(CH₂CH₂O)_(m)—CH₂CH₂NR⁷R¹⁰, —(CH₂)_(n)—C(═O)NR⁷R¹⁰,—O—(CH₂)_(m)—C(═O)NR⁷R¹⁰, —(CH₂)_(n)-(Z)_(g)—R⁷,—O—(CH₂)_(m)-(Z)_(g)—R⁷, —(CH₂)_(n)—NR¹⁰—CH₂(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸,—O—(CH₂)_(m)—NR¹⁰—CH₂(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸, —(CH₂)_(n)—CO₂R⁷,—O—(CH₂)_(m)—CO₂R⁷, —OSO₃H, —O-glucuronide, —O-glucose,

The preferred R^(L) groups include —H, —OH, —N(R⁷)₂, especially whereeach R⁷ is hydrogen.

In the alkylene chain in formula (A), it is preferred that when oneR^(L) group bonded to a carbon atoms is other than hydrogen, then theother R^(L) bonded to that carbon atom is hydrogen, i.e., the formula—CHR^(L)—. It is also preferred that at most two R^(L) groups in analkylene chain are other than hydrogen, where in the other R^(L) groupsin the chain are hydrogens. Even more preferably, only one R^(L) groupin an alkylene chain is other than hydrogen, where in the other R^(L)groups in the chain are hydrogens. In these embodiments, it ispreferable that x represents a single bond.

In another particular embodiment of the invention, all of the R^(L)groups in the alkylene chain are hydrogen. In these embodiments, thealkylene chain is represented by the formula

—(CH₂)_(o)-x-(CH₂)_(p)—.

Each R⁵ may be, independently, —(CH₂)_(m)—OR⁸, —O—(CH₂)_(m)—OR⁸,—(CH₂)_(n)—NR⁷R¹⁰, —O—(CH₂)_(m)—NR⁷R¹⁰,—(CH₂)_(n)(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸,—O—(CH₂)_(m)(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸, —(CH₂CH₂O)_(m)—R⁸,—O—(CH₂CH₂O)_(m)—R⁸, —(CH₂CH₂O)_(m)—CH₂CH₂NR⁷R¹⁰,—O—(CH₂CH₂O)_(m)—CH₂CH₂NR⁷R¹⁰, —(CH₂)_(n)—C(═O)NR⁷R¹⁰,—O—(CH₂)_(m)—C(═O)NR⁷R¹⁰, —(CH₂)_(n)-(Z)_(g)—R⁷,—O—(CH₂)_(m)-(Z)_(g)—R⁷, —(CH₂)_(n)—NR¹⁰—CH₂(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸,—O—(CH₂)_(m)—NR¹⁰—CH₂(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸, —(CH₂)_(n)—CO₂R⁷,—O—(CH₂)_(m)—CO₂R⁷, —OSO₃H, —O-glucuronide, —O-glucose,

Thus, R⁵ may be one of the following:

—(CH₂)_(m)—OR⁸,

para-(CH₂)₄—OH,

—O—(CH₂)_(m)—OR⁸,

para-O—(CH₂)₄—OH,

—(CH₂)_(n)—NR⁷R¹⁰,

para-NHSO₂CH₃,

para-CH₂NH(C═O)—(OCH₃)₃,

para-NH(C═O)CH₃,

para-CH₂NH₂,

para-NH—CO₂C₂H₅,

para-CH₂NH(C═O)CH₃,

para-CH₂NHCO₂CH₃,

para-CH₂NHSO₂CH₃,

para-(CH₂)₄—NH(C═O)O(CH₃)₃,

para-(CH₂)₄—NH₂,

para-(CH₂)₃—NH(C═O)O(CH₃)₃,

para-(CH₂)₃—NH₂,

—O—(CH₂)_(m)—NR⁷R¹⁰,

para-OCH₂CH₂NHCO₂(CH₃)₃,

para-OCH₂CH₂NHCO₂C₂H₅,

para-O—(CH₂)₃—NH—CO₂—(CH₃)₃,

para-O(CH₂)₃—NH₂,

para-OCH₂CH₂NHSO₂CH₃,

—(CH₂)_(n)(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸,

—O—(CH₂)_(m)(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸,

para-OCH₂CHOHCH₂O-glucuronide,

para-OCH₂CH₂CHOHCH₂OH,

para-OCH₂-(α-CHOH)₂CH₂OH,

para-OCH₂—(CHOH)₂CH₂OH,

—(CH₂CH₂O)_(m)—R⁸,

—O—(CH₂CH₂O)_(m)—R⁸,

—(CH₂CH₂O)_(m)—CH₂CH₂NR⁷R¹⁰,

—O—(CH₂CH₂O)_(m)—CH₂CH₂NR⁷R¹⁰,

—(CH₂)_(n)—C(═O)NR⁷R¹⁰,

para-C(═O)NH₂,

—O—(CH₂)_(m)—C(═O)NR⁷R¹⁰,

para-O—CH₂—(C═O)NHCH₂CHOH,

para-O—CH₂—(C═O)NHCH₂CHOHCH₂OH,

para-O—CH₂(C═O)NHCH₂(CHOH)₂CH₂OH,

para-O—CH₂C(C═O)NHSO₂CH₃,

para-O—CH₂(C═O)NHCO₂CH₃,

para-O—CH₂—C(C═O)NH—C(C═O)NH₂,

—O—CH₂—(C═O)NH—(C═O)CH₃,

—(CH₂)_(n)-(Z)_(g)—R⁷,

—(CH₂)_(n)—(C═N)—NH₂,

para-(C═NH)NH₂,

—(CH₂)_(n)—NH—C(═NH)—NH₂,

para-(CH₂)₃—NH—C(═NH)—NH₂,

para-CH₂NH—C(═NH)—NH₂,

—(CH₂)_(n)—CONHCH₂(CHOH)_(n)—CH₂OH,

—NH—C(═O)—CH₂—(CHOH)_(n)CH₂OH,

—NH—(C═O)—NH—CH₂(CHOH)₂CHOH,

para-NHC(C═O)NHCH₂CH₂OH,

—O—(CH₂)_(m)-(Z)_(g)—R⁷,

—O—(CH₂)_(m)—NH—C(═NH)—N(R⁷)₂,

para-O(CH₂)₃—NH—C(═NH)—NH₂,

—O—(CH₂)_(m)—CHNH₂—CO₂NR⁷R¹⁰,

para-OCH₂—CHNH₂—CO₂NH₂,

—O—(CH₂)_(m)—CHNH₂—CO₂NR⁷R¹⁰, where the compound is the (R) enantiomer,

—O—(CH₂)_(m)—CHNH₂—CO₂NR⁷R¹⁰, where the compound is the (S) enantiomer,

para-OCH₂CHOH—CH₂NHCO₂(CH₃)₃,

—(CH₂)_(n)—NR¹⁰—CH₂(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸,

para-NHCH₂(CHOH)₂CH₂OH,

—O—(CH₂)_(m)—NR¹⁰—CH₂(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸,

—O—(CH₂)_(m)—CO₂R⁷,

para-OCH₂CH₂CO₂(CH₃)₃,

para-OCH₂CO₂H,

para-OCH₂CO₂C₂H₅,

—O—(CH₂)_(m)-Boc,

—(CH₂)_(m)-Boc,

—O—(CH₂)_(m)—NH—C(═NH)—N(R⁷)₂,

—(CH₂)_(m)—NH—C(═NH)—N(R⁷)₂,

—(CH₂)_(m)—NH—C(═O)—OR⁷,

—(CH₂)_(m)—NH—C(═O)—R¹¹,

—O—(CH₂)_(m)—NH—C(═O)—R¹¹,

—O—(CH₂)_(m)—C(═O)N(R⁷)₂,

—(CH₂)_(m)—CHOH—CH₂—NHBoc,

—O—(CH₂)_(m)—CHOH—CH₂—NHBoc,

—(CH₂)_(m)—NHC(O)OR⁷,

—O—(CH₂)_(m)—NHC(O)OR⁷,

—O—(CH₂)_(m)—C(═NH)—N(R⁷)₂,

—(CH₂)_(n)—C(═NH)—N(R)₂.

In another embodiment, R⁵ is selected from the group consisting of—O—(CH₂)₃—OH, —NH₂, —O—CH₂—(CHOH)₂—CH₂OH—O—CH₂—CHOH—CH₂OH,—O—CH₂CH₂—O-tetrahydropyran-2-yl, —O—CH₂CHOH—CH₂—O-glucuronide,—O—CH₂CH₂OH, —O—(CH₂CH₂O)₄—CH₃, —O—CH₂CH₂OCH₃,—O—CH₂—(CHOC(═O)CH₃)—CH₂—OC(═O)CH₃, —O—(CH₂CH₂O)₂—CH₃,—OCH₂—CHOH—CHOH—CH₂OH, —CH₂OH, —CO₂CH₃,

In another embodiment, R⁵ is selected from the group consisting of para—O—(CH₂)₃—OH, para —NH₂, para —O—CH₂—(CHOH)₂—CH₂OH, ortho—O—CH₂—CHOH—CH₂OH, meta —O—CH₂—CHOH—CH₂OH, para—O—CH₂CH₂—O-tetrahydropyran-2-yl, para —O—CH₂CHOH—CH₂—O-glucuronide,para —O—CH₂CH₂OH, para —O—(CH₂CH₂O)₄—CH₃, para —O—CH₂CH₂OCH₃, para—O—CH₂—(CHOC(═O)CH₃)—CH₂—OC(═O)CH₃, para —O—(CH₂CH₂O)₂—CH₃,—OCH₂—CHOH—CHOH—CH₂OH, para —CH₂OH, para —CO₂CH₃, para —SO₃H, para—O-glucuronide, para

and

para

In a preferred embodiment, each —(CH₂)_(n)-(Z)_(g)—R⁷ falls within thescope of the structures described above and is, independently,

—(CH₂)_(n)(C═N)—NH₂,

—(CH₂)_(n)—NH—C(═NH)NH₂,

—(CH₂)_(n)—CONHCH₂(CHOH)_(n)—CH₂OH, or

—NH—C(═O)—CH₂—(CHOH)_(n)CH₂OH.

In another a preferred embodiment, each —O—(CH₂)_(m)-(Z)_(g)—R⁷ fallswithin the scope of the structures described above and is,independently,

—O—(CH₂)_(m)—NH—C(═NH)—N(R⁷)₂, or

—O—(CH₂)_(m)—CHNH₂—CO₂NR⁷R¹⁰.

In another preferred embodiment, R⁵ may be one of the following:

—O—CH₂CHOHCH₂O-glucuronide,

—OCH₂CHOHCH₃,

—OCH₂CH₂NH₂,

—OCH₂CH₂NHCO(CH₃)₃,

—CH₂CH₂OH,

—OCH₂CH₂OH,

—O—(CH₂)_(m)-Boc,

—(CH₂)_(m)-Boc,

—OCH₂CH₂OH,

—OCH₂CO₂H,

—O—(CH₂)_(m)—NH—C(═NH)—N(R⁷)₂,

—(CH₂)_(n)—NH—C(═NH)—N(R⁷)₂,

—NHCH₂(CHOH)₂—CH₂OH,

—OCH₂CO₂Et,

—NHSO₂CH₃,

—(CH₂)_(m)—NH—C(═O)—OR⁷,

—O—(CH₂)_(m)—NH—C(═O)—OR⁷,

—(CH₂)_(n)—NH—C(═O)—R¹¹,

—O—(CH₂)_(m)—NH—C(═O)—R¹¹,

—O—CH₂C(═O)NH₂,

—CH₂NH₂,

—NHCO₂Et,

—OCH₂CH₂CH₂CH₂OH,

—CH₂NHSO₂CH₃,

—OCH₂CH₂CHOHCH₂OH,

—OCH₂CH₂NHCO₂Et,

—NH—C(═NH2)-NH₂,

—OCH₂— (α-CHOH)₂—CH₂OH

—OCH₂CHOHCH₂NH₂,

—(CH₂)_(m)—CHOH—CH₂—NHBoc,

—O—(CH₂)_(m)—CHOH—CH₂—NHBoc,

—(CH₂)_(m)—NHC(O)OR⁷,

—O—(CH₂)_(m)—NHC(O)OR⁷,

—OCH₂CH₂CH₂NH₂,

—OCH₂CH₂NHCH₂(CHOH)₂CH₂OH,

—OCH₂CH₂NH(CH₂[(CHOH)₂CH₂OH)]₂,

—(CH₂)₄—NHBoc,

—(CH₂)₄—NH₂,

—(CH₂)₄—OH,

—OCH₂CH₂NHSO₂CH₃,

—O—(CH₂)_(m)—C(═NH)—NR⁷)₂,

—(CH₂)_(n)—C(═NH)—N(R⁷)₂,

—(CH₂)₃—NH Boc,

—(CH₂)₃NH₂,

—O—(CH₂)_(m)—NH—NH—C(═NH)—N(R⁷)₂,

—(CH₂)_(n)—NH—NH—C(═NH)—N(R⁷)₂, or

—O—CH₂—CHOH—CH₂—NH—C(═NH)—NR⁷)₂;

There are four R⁶ groups present on the ring in formula (A). Each R⁶ maybe each, independently, —R⁷, —OR¹¹, —N(R⁷)₂, —(CH₂)_(m)—OR⁸,—O—(CH₂)_(m)—OR⁸, —(CH₂)_(n)—NR⁷R¹⁰, —O—(CH₂)_(m)—NR⁷R¹⁰,—(CH₂)_(n)(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸,—O—(CH₂)_(m)(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸, —(CH₂CH₂O)_(m)—R⁸,—O—(CH₂CH₂O)_(m)—R⁸, —(CH₂CH₂O)_(m)—CH₂CH₂NR⁷R¹⁰,—O—(CH₂CH₂O)_(m)—CH₂CH₂NR⁷R¹⁰, —(CH₂)_(n)—C(═O)NR⁷R¹⁰,—O—(CH₂)_(m)—C(═O)NR⁷R¹⁰, —(CH₂)_(n)-(Z)_(g)—R⁷,—O—(CH₂)_(m)-(Z)_(g)—R⁷, —(CH₂)_(n)—NR¹⁰—CH₂(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸,—O—(CH₂)_(m)—NR¹⁰—CH₂(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸, —(CH₂)_(n)—CO₂R⁷,—O—(CH₂)_(m)—CO₂R⁷, —OSO₃H, —O-glucuronide, —O-glucose, or

In addition, one of more of the R⁶ groups can be one of the R⁵ groupswhich fall within the broad definition of R⁶ set forth above.

When two R⁶ are —OR¹¹ and are located adjacent to each other on a phenylring, the alkyl moieties of the two R⁶ groups may be bonded together toform a methylenedioxy group, i.e., a group of the formula —O—CH₂—O—.

As discussed above, R⁶ may be hydrogen. Therefore, 1, 2, 3, or 4 R⁶groups may be other than hydrogen. Preferably at most 3 of the R⁶ groupsare other than hydrogen.

Each g is, independently, an integer from 1 to 6. Therefore, each g maybe 1, 2, 3, 4, 5, or 6.

Each m is an integer from 1 to 7. Therefore, each m may be 1, 2, 3, 4,5, 6, or 7.

Each n is an integer from 0 to 7. Therefore, each n may be 0, 1, 2, 3,4, 5, 6, or 7.

Each Q in formula (A) is C—R⁵, C—R⁶, or a nitrogen atom, where at mostthree Q in a ring are nitrogen atoms. Thus, there may be 1, 2, or 3nitrogen atoms in a ring. Preferably, at most two Q are nitrogen atoms.More preferably, at most one Q is a nitrogen atom. In one particularembodiment, the nitrogen atom is at the 3-position of the ring. Inanother embodiment of the invention, each Q is either C—R⁵ or C—R⁶,i.e., there are no nitrogen atoms in the ring.

More specific examples of suitable groups represented by formula (A) areshown in formulas (B)-(E) below:

where o, x, p, R⁵, and R⁶, are as defined above;

where n is an integer from 1 to 10 and R⁵ is as defined above;

where n is an integer from 1 from 10 and R⁵ is as defined above;

where o, x, p, and R⁵ are as defined above.

In a preferred embodiment of the invention, Y is —NH₂.

In another preferred embodiment, R² is hydrogen.

In another preferred embodiment, R¹ is hydrogen.

In another preferred embodiment, X is chlorine.

In another preferred embodiment, R³ is hydrogen.

In another preferred embodiment, R^(L) is hydrogen.

In another preferred embodiment, o is 4.

In another preferred embodiment, p is 0.

In another preferred embodiment, the sum of o and p is 4.

In another preferred embodiment, x represents a single bond.

In another preferred embodiment, R⁶ is hydrogen.

In another preferred embodiment, at most one Q is a nitrogen atom.

In another preferred embodiment, no Q is a nitrogen atom.

In a preferred embodiment of the present invention:

X is halogen;

Y is —N(R⁷)₂;

R¹ is hydrogen or C₁-C₃ alkyl;

R² is —R⁷, —OR⁷, CH₂OR⁷, or —CO₂R⁷;

R³ is a group represented by formula (A); and

R⁴ is hydrogen, a group represented by formula (A), or lower alkyl;

In another preferred embodiment of the present invention:

X is chloro or bromo;

Y is —N(R⁷)₂;

R² is hydrogen or C₁-C₃ alkyl;

at most three R⁶ are other than hydrogen as described above;

at most three R^(L) are other than hydrogen as described above; and

at most 2 Q are nitrogen atoms.

In another preferred embodiment of the present invention:

Y is —NH₂;

In another preferred embodiment of the present invention:

R⁴ is hydrogen;

at most one R^(L) is other than hydrogen as described above;

at most two R⁶ are other than hydrogen as described above; and

at most 1 Q is a nitrogen atom.

The compounds of formula (I) may be prepared and used as the free base.Alternatively, the compounds may be prepared and used as apharmaceutically acceptable salt. Pharmaceutically acceptable salts aresalts that retain or enhance the desired biological activity of theparent compound and do not impart undesired toxicological effects.Examples of such salts are (a) acid addition salts formed with inorganicacids, for example, hydrochloric acid, hydrobromic acid, sulfuric acid,phosphoric acid, nitric acid and the like; (b) salts formed with organicacids such as, for example, acetic acid, oxalic acid, tartaric acid,succinic acid, maleic acid, fumaric acid, gluconic acid, citric acid,malic acid, ascorbic acid, benzoic acid, tannic acid, palmitic acid,alginic acid, polyglutamic acid, naphthalenesulfonic acid,methanesulfonic acid, p-toluenesulfonic acid, naphthalenedisulfonicacid, polygalacturonic acid, malonic acid, sulfosalicylic acid, glycolicacid, 2-hydroxy-3-naphthoate, pamoate, salicylic acid, stearic acid,phthalic acid, mandelic acid, lactic acid and the like; and (c) saltsformed from elemental anions for example, chlorine, bromine, and iodine.

It is to be noted that all enantiomers, diastereomers, and racemicmixtures of compounds within the scope of formula (I) are embraced bythe present invention. All mixtures of such enantiomers anddiastereomers are within the scope of the present invention.

Without being limited to any particular theory, it is believed that thecompounds of formula (I) function in vivo as sodium channel blockers. Byblocking epithelial sodium channels present in mucosal surfaces thecompounds of formula (I) reduce the absorption of water by the mucosalsurfaces. This effect increases the volume of protective liquids onmucosal surfaces, rebalances the system, and thus treats disease.

The present invention also provides methods of treatment that takeadvantage of the properties of the compounds of formula (I) discussedabove. Thus, subjects that may be treated by the methods of the presentinvention include, but are not limited to, patients afflicted withcystic fibrosis, primary ciliary dyskinesia, chronic bronchitis, chronicobstructive airway disease, artificially ventilated patients, patientswith acute pneumonia, etc. The present invention may be used to obtain asputum sample from a patient by administering the active compounds to atleast one lung of a patient, and then inducing or collecting a sputumsample from that patient. Typically, the invention will be administeredto respiratory mucosal surfaces via aerosol (liquid or dry powders) orlavage.

Subjects that may be treated by the method of the present invention alsoinclude patients being administered supplemental oxygen nasally (aregimen that tends to dry the airway surfaces); patients afflicted withan allergic disease or response (e.g., an allergic response to pollen,dust, animal hair or particles, insects or insect particles, etc.) thataffects nasal airway surfaces; patients afflicted with a bacterialinfection e.g., staphylococcus infections such as Staphylococcus aureusinfections, Hemophilus influenza infections, Streptococcus pneumoniaeinfections, Pseudomonas aeuriginosa infections, etc.) of the nasalairway surfaces; patients afflicted with an inflammatory disease thataffects nasal airway surfaces; or patients afflicted with sinusitis(wherein the active agent or agents are administered to promote drainageof congested mucous secretions in the sinuses by administering an amounteffective to promote drainage of congested fluid in the sinuses), orcombined, Rhinosinusitis. The invention may be administered torhino-sinal surfaces by topical delivery, including aerosols and drops.

The present invention may be used to hydrate mucosal surfaces other thanairway surfaces. Such other mucosal surfaces include gastrointestinalsurfaces, oral surfaces, genito-urethral surfaces, ocular surfaces orsurfaces of the eye, the inner ear and the middle ear. For example, theactive compounds of the present invention may be administered by anysuitable means, including locally/topically, orally, or rectally, in aneffective amount.

The compounds of the present invention are also useful for treating avariety of functions relating to the cardiovascular system. Thus, thecompounds of the present invention are useful for use asantihypertensive agents. The compounds may also be used to reduce bloodpressure and to treat edema. In addition, the compounds of the presentinvention are also useful for promoting diuresis, natriuresis, andsaluresis. The compounds may be used alone or in combination with betablockers, ACE inhibitors, HMGCoA reductase inhibitors, calcium channelblockers and other cardiovascular agents to treat hypertension,congestive heart failure and reduce cardiovascular mortality.

The present invention is concerned primarily with the treatment of humansubjects, but may also be employed for the treatment of other mammaliansubjects, such as dogs and cats, for veterinary purposes.

As discussed above, the compounds used to prepare the compositions ofthe present invention may be in the form of a pharmaceuticallyacceptable free base. Because the free base of the compound is generallyless soluble in aqueous solutions than the salt, free base compositionsare employed to provide more sustained release of active agent to thelungs. An active agent present in the lungs in particulate form whichhas not dissolved into solution is not available to induce aphysiological response, but serves as a depot of bioavailable drug whichgradually dissolves into solution.

Another aspect of the present invention is a pharmaceutical composition,comprising a compound of formula (I) in a pharmaceutically acceptablecarrier (e.g., an aqueous carrier solution). In general, the compound offormula (I) is included in the composition in an amount effective toinhibit the reabsorption of water by mucosal surfaces.

The compounds of the present invention may also be used in conjunctionwith a P2Y2 receptor agonist or a pharmaceutically acceptable saltthereof (also sometimes referred to as an “active agent” herein). Thecomposition may further comprise a P2Y2 receptor agonist or apharmaceutically acceptable salt thereof (also sometimes referred to asan “active agent” herein). The P2Y2 receptor agonist is typicallyincluded in an amount effective to stimulate chloride and watersecretion by airway surfaces, particularly nasal airway surfaces.Suitable P2Y2 receptor agonists are described in columns 9-10 of U.S.Pat. No. 6,264,975, U.S. Pat. No. 5,656,256, and U.S. Pat. No.5,292,498, each of which is incorporated herein by reference.

Bronchodiloators can also be used in combination with compounds of thepresent invention. These bronchodilators include, but are not limitedto, β-adrenergic agonists including but not limited to epinephrine,isoproterenol, fenoterol, albutereol, terbutalin, pirbuterol,bitolterol, metaproterenol, iosetharine, salmeterol xinafoate, as wellas anticholinergic agents including but not limited to ipratropiumbromide, as well as compounds such as theophylline and aminophylline.These compounds may be administered in accordance with known techniques,either prior to or concurrently with the active compounds describedherein.

Another aspect of the present invention is a pharmaceutical formulation,comprising an active compound as described above in a pharmaceuticallyacceptable carrier (e.g., an aqueous carrier solution). In general, theactive compound is included in the composition in an amount effective totreat mucosal surfaces, such as inhibiting the reabsorption of water bymucosal surfaces, including airway and other surfaces.

The active compounds disclosed herein may be administered to mucosalsurfaces by any suitable means, including topically, orally, rectally,vaginally, ocularly and dermally, etc. For example, for the treatment ofconstipation, the active compounds may be administered orally orrectally to the gastrointestinal mucosal surface. The active compoundmay be combined with a pharmaceutically acceptable carrier in anysuitable form, such as sterile physiological or dilute saline or topicalsolution, as a droplet, tablet or the like for oral administration, as asuppository for rectal or genito-urethral administration, etc.Excipients may be included in the formulation to enhance the solubilityof the active compounds, as desired.

The active compounds disclosed herein may be administered to the airwaysurfaces of a patient by any suitable means, including as a spray, mist,or droplets of the active compounds in a pharmaceutically acceptablecarrier such as physiological or dilute saline solutions or distilledwater. For example, the active compounds may be prepared as formulationsand administered as described in U.S. Pat. No. 5,789,391 to Jacobus, thedisclosure of which is incorporated by reference herein in its entirety.

Solid or liquid particulate active agents prepared for practicing thepresent invention could, as noted above, include particles of respirableor non-respirable size; that is, for respirable particles, particles ofa size sufficiently small to pass through the mouth and larynx uponinhalation and into the bronchi and alveoli of the lungs, and fornon-respirable particles, particles sufficiently large to be retained inthe nasal airway passages rather than pass through the larynx and intothe bronchi and alveoli of the lungs. In general, particles ranging fromabout 1 to 5 microns in size (more particularly, less than about 4.7microns in size) are respirable. Particles of non-respirable size aregreater than about 5 microns in size, up to the size of visibledroplets. Thus, for nasal administration, a particle size in the rangeof 10-500 μm may be used to ensure retention in the nasal cavity.

In the manufacture of a formulation according to the invention, activeagents or the physiologically acceptable salts or free bases thereof aretypically admixed with, inter alia, an acceptable carrier. Of course,the carrier must be compatible with any other ingredients in theformulation and must not be deleterious to the patient. The carrier mustbe solid or liquid, or both, and is preferably formulated with thecompound as a unit-dose formulation, for example, a capsule, that maycontain 0.5% to 99% by weight of the active compound. One or more activecompounds may be incorporated in the formulations of the invention,which formulations may be prepared by any of the well-known techniquesof pharmacy consisting essentially of admixing the components.

Compositions containing respirable or non-respirable dry particles ofmicronized active agent may be prepared by grinding the dry active agentwith a mortar and pestle, and then passing the micronized compositionthrough a 400 mesh screen to break up or separate out largeagglomerates.

The particulate active agent composition may optionally contain adispersant which serves to facilitate the formulation of an aerosol. Asuitable dispersant is lactose, which may be blended with the activeagent in any suitable ratio (e.g., a 1 to 1 ratio by weight).

Active compounds disclosed herein may be administered to airway surfacesincluding the nasal passages, sinuses and lungs of a subject by asuitable means know in the art, such as by nose drops, mists, etc. Inone embodiment of the invention, the active compounds of the presentinvention and administered by transbronchoscopic lavage. In a preferredembodiment of the invention, the active compounds of the presentinvention are deposited on lung airway surfaces by administering anaerosol suspension of respirable particles comprised of the activecompound, which the subject inhales. The respirable particles may beliquid or solid. Numerous inhalers for administering aerosol particlesto the lungs of a subject are known.

Inhalers such as those developed by Inhale Therapeutic Systems, PaloAlto, Calif., USA, may be employed, including but not limited to thosedisclosed in U.S. Pat. Nos. 5,740,794; 5,654,007; 5,458,135; 5,775,320;and 5,785,049, each of which is incorporated herein by reference. TheApplicant specifically intends that the disclosures of all patentreferences cited herein be incorporated by reference herein in theirentirety. Inhalers such as those developed by Dura Pharmaceuticals,Inc., San Diego, Calif., USA, may also be employed, including but notlimited to those disclosed in U.S. Pat. Nos. 5,622,166; 5,577,497;5,645,051; and 5,492,112, each of which is incorporated herein byreference. Additionally, inhalers such as those developed by AradigmCorp., Hayward, Calif., USA, may be employed, including but not limitedto those disclosed in U.S. Pat. Nos. 5,826,570; 5,813,397; 5,819,726;and 5,655,516, each of which is incorporated herein by reference. Theseapparatuses are particularly suitable as dry particle inhalers.

Aerosols of liquid particles comprising the active compound may beproduced by any suitable means, such as with a pressure-driven aerosolnebulizer or an ultrasonic nebulizer. See, e.g., U.S. Pat. No.4,501,729, which is incorporated herein by reference. Nebulizers arecommercially available devices which transform solutions or suspensionsof the active ingredient into a therapeutic aerosol mist either by meansof acceleration of compressed gas, typically air or oxygen, through anarrow venturi orifice or by means of ultrasonic agitation. Suitableformulations for use in nebulizers consist of the active ingredient in aliquid carrier, the active ingredient comprising up to 40% w/w of theformulation, but preferably less than 20% w/w. The carrier is typicallywater (and most preferably sterile, pyrogen-free water) or diluteaqueous alcoholic solution. Perfluorocarbon carriers may also be used.Optional additives include preservatives if the formulation is not madesterile, for example, methyl hydroxybenzoate, antioxidants, flavoringagents; volatile oils, buffering agents and surfactants.

Aerosols of solid particles comprising the active compound may likewisebe produced with any solid particulate medicament aerosol generator.Aerosol generators for administering solid particulate medicaments to asubject produce particles which are respirable, as explained above, andgenerate a volume of aerosol containing predetermined metered dose ofmedicament at a rate suitable for human administration. One illustrativetype of solid particulate aerosol generator is an insufflator. Suitableformulations for administration by insufflation include finelycomminuted powders which may be delivered by means of an insufflator ortaken into the nasal cavity in the manner of a snuff. In theinsufflator, the powder (e.g., a metered dose thereof effective to carryout the treatments described herein) is contained in capsules orcartridges, typically made of gelatin or plastic, which are eitherpierced or opened in situ and the powder delivered by air drawn throughthe device upon inhalation or by means of a manually-operated pump. Thepowder employed in the insufflator consists either solely of the activeingredient or of powder blend comprising the active ingredient, asuitable powder diluent, such as lactose, and an optional surfactant.The active ingredient typically comprises of 0.1 to 100% w/w of theformulation. A second type of illustrative aerosol generator comprises ametered dose inhaler. Metered dose inhalers are pressurized aerosoldispensers, typically containing a suspension or solution formulation ofactive ingredient in a liquified propellant. During use, these devicesdischarge the formulation through a valve adapted to deliver a meteredvolume, typically from 10 to 150 to produce a fine particle spraycontaining the active ingredient. Suitable propellants include certainchlorofluorocarbon compounds, for example, dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane and mixtures thereof.The formulation may additionally contain one of more co-solvents, forexample, ethanol, surfactants, such as oleic acid or sorbitan trioleate,antioxidants and suitable flavoring agents.

The aerosol, whether formed from solid or liquid particles, may beproduced by the aerosol generator at a rate of from about 10 to 150liters per minute, more preferable from 30 to 150 liters per minute, andmost preferably about 60 liters per minute. Aerosols containing greateramounts of medicament may be administered more rapidly.

The dosage of the active compounds disclosed herein will vary dependingon the condition being treated and the state of the subject, butgenerally may be from about 0.01, 0.03, 0.05, 0.1 to 1, 5, 10 or 20 mgof the pharmaceutic agent, deposited on the airway surfaces. The dailydose may be divided among one or multiple unit dose administrations. Thegoal is to achieve a concentration of the pharmaceutic agents on lungairway surfaces of between 10⁻⁹-10⁴ M.

In another embodiment, they are administered by administering an aerosolsuspension of respirable or non-respirable particles (preferablynon-respirable particles) comprised of active compound, which thesubject inhales through the nose. The respirable or non-respirableparticles may be liquid or solid. The quantity of active agent includedmay be an amount of sufficient to achieve dissolved concentrations ofactive agent on the airway surfaces of the subject of from about 10⁻⁹,10⁻⁸, or 10⁻⁷ to about 10⁻³, 10⁻², 10⁻¹ moles/liter, and more preferablyfrom about 10⁻⁹ to about 10⁻⁴ moles/liter.

The dosage of active compound will vary depending on the condition beingtreated and the state of the subject, but generally may be an amountsufficient to achieve dissolved concentrations of active compound on thenasal airway surfaces of the subject from about 10⁻⁹, 10⁻⁸, 10⁻⁷ toabout 10⁻³, 10⁻², or 10⁻¹ moles/liter, and more preferably from about10⁻⁷ to about 10⁻⁴ moles/liter. Depending upon the solubility of theparticular formulation of active compound administered, the daily dosemay be divided among one or several unit dose administrations. The dailydose by weight may range from about 0.01, 0.03, 0.1, 0.5 or 1.0 to 10 or20 milligrams of active agent particles for a human subject, dependingupon the age and condition of the subject. A currently preferred unitdose is about 0.5 milligrams of active agent given at a regimen of 2-10administrations per day. The dosage may be provided as a prepackagedunit by any suitable means (e.g., encapsulating a gelatin capsule).

In one embodiment of the invention, the particulate active agentcomposition may contain both a free base of active agent and apharmaceutically acceptable salt to provide both early release andsustained release of active agent for dissolution into the mucussecretions of the nose. Such a composition serves to provide both earlyrelief to the patient, and sustained relief over time. Sustained relief,by decreasing the number of daily administrations required, is expectedto increase patient compliance with the course of active agenttreatments.

Pharmaceutical formulations suitable for airway administration includeformulations of solutions, emulsions, suspensions and extracts. Seegenerally, J. Nairn, Solutions, Emulsions, Suspensions and Extracts, inRemington: The Science and Practice of Pharmacy, chap. 86 (19^(th) ed.1995), incorporated herein by reference. Pharmaceutical formulationssuitable for nasal administration may be prepared as described in U.S.Pat. Nos. 4,389,393 to Schor; 5,707,644 to Mum; 4,294,829 to Suzuki; and4,835,142 to Suzuki, the disclosures of which are incorporated byreference herein in their entirety.

Mists or aerosols of liquid particles comprising the active compound maybe produced by any suitable means, such as by a simple nasal spray withthe active agent in an aqueous pharmaceutically acceptable carrier, suchas a sterile saline solution or sterile water. Administration may bewith a pressure-driven aerosol nebulizer or an ultrasonic nebulizer. Seee.g. U.S. Pat. Nos. 4,501,729 and 5,656,256, both of which areincorporated herein by reference. Suitable formulations for use in anasal droplet or spray bottle or in nebulizers consist of the activeingredient in a liquid carrier, the active ingredient comprising up to40% w/w of the formulation, but preferably less than 20% w/w. Typicallythe carrier is water (and most preferably sterile, pyrogen-free water)or dilute aqueous alcoholic solution, preferably made in a 0.12% to 0.8%solution of sodium chloride. Optional additives include preservatives ifthe formulation is not made sterile, for example, methylhydroxybenzoate, antioxidants, flavoring agents, volatile oils,buffering agents, osmotically active agents (e.g. mannitol, xylitol,erythritol) and surfactants.

Compositions containing respirable or non-respirable dry particles ofmicronized active agent may be prepared by grinding the dry active agentwith a mortar and pestle, and then passing the micronized compositionthrough a 400 mesh screen to break up or separate out largeagglomerates.

The particulate composition may optionally contain a dispersant whichserves to facilitate the formation of an aerosol. A suitable dispersantis lactose, which may be blended with the active agent in any suitableratio (e.g., a 1 to 1 ratio by weight).

The compounds of formula (I) may be synthesized according to proceduresknown in the art. A representative synthetic procedure is shown in thescheme below:

These procedures are described in, for example, E. J. Cragoe, “TheSynthesis of Amiloride and Its Analogs” (Chapter 3) in Amiloride and ItsAnalogs, pp. 25-36, incorporated herein by reference. Other methods ofpreparing the compounds are described in, for example, U.S. Pat. No.3,313,813, incorporated herein by reference. See in particular MethodsA, B, C, and D described in U.S. Pat. No. 3,313,813. Several assays maybe used to characterize the compounds of the present invention.Representative assays are discussed below.

In Vitro Measure of Sodium Channel Blocking Activity and Reversibility

One assay used to assess mechanism of action and/or potency of thecompounds of the present invention involves the determination of lumenaldrug inhibition of airway epithelial sodium currents measured undershort circuit current (I_(SC)) using airway epithelial monolayersmounted in Using chambers. Cells obtained from freshly excised human,dog, sheep or rodent airways are seeded onto porous 0.4 micron Snapwell™Inserts (CoStar), cultured at air-liquid interface (ALI) conditions inhormonally defined media, and assayed for sodium transport activity(I_(SC)) while bathed in Krebs Bicarbonate Ringer (KBR) in Usingchambers. All test drug additions are to the lumenal bath with half-logdose addition protocols (from 1×10⁻¹¹ M to 3×10⁻⁵ M), and the cumulativechange in I_(SC) (inhibition) recorded. All drugs are prepared indimethyl sulfoxide as stock solutions at a concentration of 1×10⁻² M andstored at −20° C. Eight preparations are typically run in parallel; twopreparations per run incorporate amiloride and/or benzamil as positivecontrols. After the maximal concentration (5×10⁻⁵ M) is administered,the lumenal bath is exchanged three times with fresh drug-free KBRsolution, and the resultant I_(SC) measured after each wash forapproximately 5 minutes in duration. Reversibility is defined as thepercent return to the baseline value for sodium current after the thirdwash. All data from the voltage clamps are collected via a computerinterface and analyzed off-line.

Dose-effect relationships for all compounds are considered and analyzedby the Prism 3.0 program. IC₅₀ values, maximal effective concentrations,and reversibility are calculated and compared to amiloride and benzamilas positive controls.

Pharmacological Assays of Absorption

(1) Apical Disappearance Assay

Bronchial cells (dog, human, sheep, or rodent cells) are seeded at adensity of 0.25×10⁶/cm² on a porous Transwell-Col collagen-coatedmembrane with a growth area of 1.13 cm² grown at an air-liquid interfacein hormonally defined media that promotes a polarized epithelium. From12 to 20 days after development of an air-liquid interface (ALI) thecultures are expected to be >90% ciliated, and mucins will accumulate onthe cells. To ensure the integrity of primary airway epithelial cellpreparations, the transepithelial resistance (R_(t)) and transepithelialpotential differences (PD), which are indicators of the integrity ofpolarized nature of the culture, are measured. Human cell systems arepreferred for studies of rates of absorption from apical surfaces. Thedisappearance assay is conducted under conditions that mimic the “thin”films in vivo (˜25 μl) and is initiated by adding experimental sodiumchannel blockers or positive controls (amiloride, benzamil, phenamil) tothe apical surface at an initial concentration of 10 μM. A series ofsamples (5 μl volume per sample) is collected at various time points,including 0, 5, 20, 40, 90 and 240 minutes. Concentrations aredetermined by measuring intrinsic fluorescence of each sodium channelblocker using a Fluorocount Microplate Fluorometer or HPLC. Quantitativeanalysis employs a standard curve generated from authentic referencestandard materials of known concentration and purity. Data analysis ofthe rate of disappearance is performed using nonlinear regression, onephase exponential decay (Prism V 3.0).

2. Confocal Microscopy Assay of Amiloride Congener Uptake

Virtually all amiloride-like molecules fluoresce in the ultravioletrange. This property of these molecules may be used to directly measurecellular update using x-z confocal microscopy. Equimolar concentrationsof experimental compounds and positive controls including amiloride andcompounds that demonstrate rapid uptake into the cellular compartment(benzamil and phenamil) are placed on the apical surface of airwaycultures on the stage of the confocal microscope. Serial x-z images areobtained with time and the magnitude of fluorescence accumulating in thecellular compartment is quantitated and plotted as a change influorescence versus time.

3. In Vitro Assays of Compound Metabolism

Airway epithelial cells have the capacity to metabolize drugs during theprocess of transepithelial absorption. Further, although less likely, itis possible that drugs can be metabolized on airway epithelial surfacesby specific ectoenzyme activities. Perhaps more likely as anecto-surface event, compounds may be metabolized by the infectedsecretions that occupy the airway lumens of patients with lung disease,e.g. cystic fibrosis. Thus, a series of assays is performed tocharacterize the compound metabolism that results from the interactionof test compounds with human airway epithelia and/or human airwayepithelial lumenal products.

In the first series of assays, the interaction of test compounds in KBRas an “ASL” stimulant are applied to the apical surface of human airwayepithelial cells grown in the T-Col insert system. For most compounds,metabolism (generation of new species) is tested for using highperformance liquid chromatography (HPLC) to resolve chemical species andthe endogenous fluorescence properties of these compounds to estimatethe relative quantities of test compound and novel metabolites. For atypical assay, a test solution (25 μl KBR, containing 10 μM testcompound) is placed on the epithelial lumenal surface. Sequential 5 to10 μl samples are obtained from the lumenal and serosal compartments forHPLC analysis of (1) the mass of test compound permeating from thelumenal to serosal bath and (2) the potential formation of metabolitesfrom the parent compound. In instances where the fluorescence propertiesof the test molecule are not adequate for such characterizations,radiolabeled compounds are used for these assays. From the HPLC data,the rate of disappearance and/or formation of novel metabolite compoundson the lumenal surface and the appearance of test compound and/or novelmetabolite in the basolateral solution is quantitated. The data relatingthe chromatographic mobility of potential novel metabolites withreference to the parent compound are also quantitated.

To analyze the potential metabolism of test compounds by CF sputum, a“representative” mixture of expectorated CF sputum obtained from 10 CFpatients (under IRB approval) has been collected. The sputum has been besolubilized in a 1:5 mixture of KBR solution with vigorous vortexing,following which the mixture was split into a “neat” sputum aliquot andan aliquot subjected to ultracentrifugation so that a “supernatant”aliquot was obtained (neat=cellular; supernatant=liquid phase). Typicalstudies of compound metabolism by CF sputum involve the addition ofknown masses of test compound to “neat” CF sputum and aliquots of CFsputum “supernatant” incubated at 37° C., followed by sequentialsampling of aliquots from each sputum type for characterization ofcompound stability/metabolism by HPLC analysis as described above. Asabove, analysis of compound disappearance, rates of formation of novelmetabolities, and HPLC mobilities of novel metabolites are thenperformed.

4. Pharmacological Effects and Mechanism of Action of the Drug inAnimals

The effect of compounds for enhancing mucociliary clearance (MCC) can bemeasured using an in vivo model described by Sabater et al., Journal ofApplied Physiology, 1999, pp. 2191-2196, incorporated herein byreference.

EXAMPLES

Having generally described this invention, a further understanding canbe obtained by reference to certain specific examples which are providedherein for purposes of illustration only and are not intended to belimiting unless otherwise specified.

Preparation of Sodium Channel Blockers

Materials and methods. All reagents and solvents were purchased fromAldrich Chemical Corp. and used without further purification. NMRspectra were obtained on either a Bruker WM 360 (¹H NMR at 360 MHz and¹³C NMR at 90 MHz) or a Bruker AC 300 (¹H NMR at 300 MHz and ¹³C NMR at75 MHz). Flash chromatography was performed on a Flash Elute™ systemfrom Elution Solution (PO Box 5147, Charlottesville, Va. 22905) chargedwith a 90 g silica gel cartridge (40M FSO-0110-040155, 32-63 μm) at 20psi (N₂).

GC-analysis was performed on a Shimadzu GC-17 equipped with a HeliflexCapillary Column (Alltech); Phase: AT-1, Length: 10 meters, ID: 0.53 mm,Film: 0.25 micrometers. GC Parameters: Injector at 320° C., Detector at320° C., FID gas flow: H₂ at 40 ml/min., Air at 400 ml/min. Carrier gas:Split Ratio 16:1, N₂ flow at 15 ml/min., N₂ velocity at 18 cm/sec. Thetemperature program is 70° C. for 0-3 min, 70-300° C. from 3-10 min,300° C. from 10-15 min.

HPLC analysis was performed on a Gilson 322 Pump, detector UV/Vis-156 at360 nm, equipped with a Microsorb MV C8 column, 100 A, 25 cm. Mobilephase: A=acetonitrile with 0.1% TFA, B=water with 0.1% TFA. Gradientprogram: 95:5 B:A for 1 min, then to 20:80 B:A over 7 min, then to 100%A over 1 min, followed by washout with 100% A for 11 min, flow rate: 1ml/min.

Example 14-(4-Carboxymethylphenyl)butylamidino-3,5-diamino-6-chloropyrazinecarboxamidehydrochloride (9)

Methanesulfonic acid 4-(4-carboxymethylphenyl)butyl ester (6)

Compound 6 was prepared according to the published procedure¹. ¹H NMR(300 MHz, CDCl₃) δ 1.75 (m, 4H), 2.78 (m, 2H), 3.12 (s, 3H), 3.88 (s,3H), 4.22 (m, 2H), 7.28 (d, 2H), 7.98 (d, 2H)

4-(4-Carboxymethylphenyl)butylazide (7). Typical Procedure C

Compound 6 (6 g, 0.02 mol) was dissolved in 80 ml of dry DMF then sodiumazide (1.8 g, 0.027 mol) was added. The suspension was stirred at 80° C.(oil bath) for 3 h. The solvent was then removed at reduced pressure andthe residual oil was treated with CH₂Cl₂ (100 mL).

The resulting solution was washed with water (2×100 mL), brine and driedover magnesium sulfate. The solvent was removed under reduced pressurethen the residue was redissolved in a 1:1 mixture of ethylacetate/hexanes (200 mL) and passed through a pad of silica gel. Thesolvent was removed under reduced pressure to give 4.1 g (85%) of 7 asclear oil. ¹H NMR (300 MHz, CDCl₃) δ 1.68 (m, 4H), 2.22 (t, 2H), 3.29(t, 3H), 3.92 (s, 3H), 7.28 (d, 2H), 7.98 (d, 2H).

4-(4-Carboxymethylphenyl)butylamine (8)

Azide 7 (1.7 g, 7.2 mmol) and triphenylphosphine (1.9 g, 7.2 mmol) weredissolved in a 10% solution of water in THF (66 mL) and stirredovernight at 25° C. Then more triphenylphosphine (0.8 g, 3 mmol) wasadded and the heating was continued at 60° C. (oil bath) for 6 h. Thesolvent was removed under reduced pressure and the residue was treatedwith 2M HCl (100 mL) and extracted with ethyl acetate (2×50 mL). Thewater fraction was collected and ammonium hydroxide was added until thepH reached approximately 13. The mixture was extracted with ethylacetate (2×100 mL) then the organic fraction was washed with brine,water and dried with sodium sulfate. Ethyl acetate was removed underreduced pressure to give 0.8 g (53%) of amine 8.

4-(4-Carboxymethylphenyl)butylamidino-3,5-diamino-6-chloropyrazinecarboxamidehydrochloride (9). Typical Procedure D

1-(3,5-Diamino-6-chloropyrazinoyl-2-methyl-pseudothiourea hydroiodide(0.2 g, 0.5 mmol) was added to a solution of 8 (0.7 g, 3.4 mmol) in THF(20 mL). The reaction mixture was stirred at reflux for 6 h, then thesolvent was evaporated and the resultant oil was treated with 10% HCl(15 mL). The precipitate was isolated and crystallized twice fromethanol to give 9 (53 mg, 25%) as a yellow solid. ¹H NMR (300 MHz,DMSO-d₆) δ1.59 (br s, 4H), 2.71 (m, 2H), 3.83 (s, 3H), 7.40 (d, 2H),7.48 (br s, 2H), 7.80 (d, 2H), 8.92 (br s, 2H), 9.00 (br s, 1H), 9.48(br s, 2H), 10.55 (s, 1H). APCI MS m/z=420 [C₁₈H₂₂ClN₇O₃+H]⁺.

Example 24-(4-Sulfatephenyl)butylamidino-3,5-diamino-6-chloropyrazinecarboxamide(10)

4-(4-Sulfatephenyl)butylamidino-3,5-diamino-6-chloropyrazinecarboxamide(10)

4-(4-Hydroxyphenyl)butylamidino-3,5-diamino-6-chloropyrazinecarboxamidehydrochloride (0.2 g, 0.5 mmol) was dissolved in 5 mL of dry pyridineand pyridine sulfurtrioxide (450 mg, 2.5 mmol) was added. The reactionmixture was stirred overnight at room temperature and the precipitatethat formed was isolated by filtration and washed with ethyl acetate(2×25 mL) to give crude 10 (180 mg, 39%, purity 87% by HPLC). An aliquotof the crude 10 (67 mg) was purified by flash chromatography (silicagel, 6:3:0.1 methylene chloride/methanol/concentrated ammoniumhydroxide) to give 10 as a yellow solid (9.3 mg, 4% based on starting5). ¹H NMR (300 MHz, DMSO-d₆) δ 1.59 (br s, 4H), 2.58 (m, 2H), 3.28 (m,2H), 7.08 (s, 4H), 7.1-7.9 (m, 6H). ESI MS m/z=456 [C₁₆H₂₀ClN₇O₆S—H]⁻.

Example 34-[4-(2,3-Dihydroxypropyloxyl)phenyl]butylamidino-3,5-diamino-6-chloropyrazinecarboxamidehydrochloride (33)

N-Cbz-4-(4-hydroxyphenyl)butylamine (29)

To vigorously stirred suspension of 4 (10.5 g, 0.043 mol) in THF(approx. 150 mL) was added sodium hydrogencarbonate (11 g, 0.13 mol) andthen water until a clear solution was obtained (approx. 50 mL). Thereaction mixture was cooled to 0° C. then benzyl chloroformate (10 mL,0.07 mol) was added and the reaction was stirred overnight. The solventwas removed at reduced pressure then ethyl acetate (approx. 100 mL) wasadded to the residue. The organics were washed with HCl (2 M solution,2×30 mL), water (2×50 mL), and dried over sodium sulfate. The solventwas removed and the residue was purified by column chromatography(silica gel, 1:1 ethyl acetate/hexanes) to provide 29 (10 g, 85%) as awhite solid. ¹H NMR (300 MHz, CDCl₃) δ 1.55 (br s, 4H), 2.53 (m, 2H),3.19 (m, 2H), 5.05 (s, 2H), 5.83 (s, 1H), 6.73 (d, 2H), 7.00 (d, 2H),7.38 (m, 5H).

N-Cbz-4-(4-allyloxyphenyl)butylamine (30)

Potassium tert-butoxide (1.7 g, 15.2 mmol) and 18-crown-6 (0.1 g, 0.3mmol) were added to a solution of 29 (4.3 g, 14.3 mmol) in dry MeCN (80mL) and the mixture was stirred for 20 min at room temperature. Afterthis time, allyl bromide (1.2 mL, 14.3 mmol) in MeCN (10 mL) was added.The reaction mixture was stirred overnight at room temperature, then theprecipitate was filtered off and washed with ethyl acetate. The organicfractions were combined, the solvent was removed at reduced pressure andthe residue was purified twice by flash chromatography (silica gel, 1:1ethyl acetate/hexanes) to provide compound 30 (3.4 g, 71%) as a whitesolid. ¹H NMR (300 MHz, CDCl₃) δ 1.55 (m, 4H), 2.54 (t, 2H), 3.20 (m,2H), 4.50 (d, 2H), 5.08 (s, 2H), 5.28 (d, 1H), 5.40 (d, 1H), 6.06 (m,1H), 6.82 (d, 2H), 7.05 (d, 2H), 7.33 (s, 5H)

N-Cbz-4-[(2,3-dihydroxypropyloxy)phenyl]butylamine (31)

A solution of osmium tetroxide (50 mg, 0.2 mmol) in tert-butanol (8 mL)was added to a solution of 4-methylmorpholine N-oxide monohydrate (1.2g, 9.1 mmol) in 100 mL (1:1) acetone/water solution and the mixture wasstirred for 10 min at room temperature. After this time, 30 (3.1 g, 9.0mmol) was added in 50 mL (1:1) acetone/water solution. The reactionmixture was stirred at room temperature overnight, then NaHSO₃ (0.5 g)was added and the stirring was continued for 15 min. The acetone wasevaporated and the pH was adjusted to 5.5 by the addition of 2N HCl thenthe mixture was extracted with ethyl acetate. The organic fraction wasisolated, dried with sodium sulfate, and filtered through silica gel.Compound 31 (2.1 g, 62%) was isolated as a white solid after removingthe solvent and drying under vacuum. ¹H NMR (300 MHz, DMSO-d₆) δ 1.48(m, 2H), 1.50 (m, 2H), 2.46 (m, 2H), 3.00 (m, 2H), 3.43 (m, 2H), 3.80(m, 2H), 3.93 (t, 1H), 4.66 (d, 1H), 4.99 (s, 2H), 6.82 (d, 2H), 7.07(d, 2H), 7.26 (s, 1H), 7.33 (s, 5H).

4-[(2,3-Dihydroxypropyloxy)phenyl]butylamine (32)

Cbz-protected amine 31 (2.1 g, 5.6 mmol) was dissolved in methanol (50mL) and Pd/C (0.46 g, 5% wet) was added in methanol (20 mL). Thereaction mixture was stirred for 3 h at 1 atmosphere of hydrogen, thenthe solution was filtered through a pad of silica gel. The solvent wasthen evaporated to give free amine 32 (0.9 g, 66%). ¹H NMR (300 MHz,DMSO-d₆) δ 1.37 (m, 2H), 1.50 (m, 2H), 2.92 (m, 1H), 3.22-4.05 (br s,4H), 3.43 (m, 2H), 3.93 (m, 1H), 6.82 (d, 2H), 7.07 (d, 2H).

4-[4-(2,3-Dihydroxypropyloxyl)phenyl]butylamidino-3,5-diamino-6-chloropyrazinecarboxamidehydrochloride (33). (General Procedure Z)

1-(3,5-Diamino-6-chloropyrazinoyl-2-methyl-pseudothiourea hydroiodide(1.5 g, 3.8 mmol) was added to a solution of 32 (0.9 g, 3.7 mmol) in amixture of THF (50 mL) and diisopropylethylamine (2 mL). The reactionmixture was stirred at reflux (66° C.) for 4 h. After this time, thereaction mixture was cooled to room temperature and the formedprecipitate was isolated as a yellow solid. The obtained solid waswashed with 5% HCl, water, and dried under vacuum to give 33 (0.88 g) asa yellow solid. The mother liquor was evaporated and the residue waspurified by flash chromatography (silica gel, 5:1:05chloroform/methanol/concentrated ammonium hydroxide). The isolated freeamino compound was treated with 5% HCl to give an additional portion of33 (0.12 g). The total yield of 33 was 53%. ¹H NMR (300 MHz, DMSO-d₆) δ1.56 (m, 4H), 2.56 (br s, 2H), 3.31 (m, 2H), 3.42 (m, 2H), 3.82 (m, 2H),3.93 (m, 2H), 4.32 (br s, 4H), 6.84 (d, 2H), 7.10 (d, 2H), 7.45 (br s,2H), 8.81 (br s, 1H), 8.94 (br s, 1H), 9.25 (br s, 1H), 10.52 (s, 1H).APCI MS m/z=452 [C₁₉H₂₆ClN₇O₄+H]⁺.

Example 4 Substituted 3,5-diamino-6-chloropyrazinecarboxamide amidines(cont) Alternate Preparation of amine 32 4-(4-Methoxyphenyl)butyramide(117)

4-(4-Methoxyphenyl)butyric acid (1) (450 g, 2.32 mole) was combined withdry THF (4 L) and 4-methylmorpholine (268 mL, 2.43 mole) in a 12 L threeneck flask with mechanical stirring, an ice-methanol cooling bath andnitrogen atmosphere. Small pieces of dry ice were used to bring the bathtemperature below −20° C. Isobutyl chloroformate was added at a rate soas not to exceed an internal temperature of −10° C. After stirring for30 min. at −10 to −20° C., a 4.7 M solution of ammonia in methanol (990mL, 4.64 mole) was added in one portion. During the addition, thereaction temperature rose to 0° C. The reaction was allowed to stir for30 min. and then allowed to stand overnight. The product mixture wastransferred to a 22 L separatory funnel with ethyl acetate (6 L), and10% sodium chloride solution (1.5 L). The layers were separated and theorganic solution was washed with 10% sodium chloride solution (4×1 L)and then brine (3×500 mL). The organic layer was dried over sodiumsulfate, filtered, evaporated and placed under high vacuum overnight.This afforded 432 g (97%) of the pure amide (117) as an off white solid.

¹H NMR (300 MHz, CD₃OD) δ 1.81-1.93 (m, 2H), 2.20 (t, J=7.7 Hz, 2H),2.57 (t, J=7.7 Hz, 2H), 3.74 (s, 3H), 6.82 (d, J=8.7 Hz), 7.09 (d, J=8.7Hz, 1H). CI MS m/z=194 [C₁₁H₁₅NO₂+H]⁺.

4-(4-Hydroxyphenyl)butylamine hydrobromide (4)

4-(4-Methoxyphenyl)butyramide (117) (200 g, 1.0 mole) and THF (300 mL)were combined in a 12 L three neck flask which was equipped with aheating mantle, an internal thermometer and a reflux condenser. Thesuspension was slowly mechanically stirred while a 1 M borane-THEcomplex (1 L, 1 mole) was dripped in via a pressure equalizing additionfunnel over 20 min. Another 2.2 L (2.2 mole) of 1 M borane.THF complexwas dripped in over 20 min. The reaction temperature rose to 45° C.during the addition. The reaction was stirred and heated to reflux over1 h, at reflux for 2 h and then allowed to cool for 2 h. Methanol (500mL) was slowly and cautiously dripped into the reaction. Copious H₂evolution was observed. The reaction was heated at reflux for 2 h andallowed to cool overnight. The reaction was evaporated and thenco-evaporated with toluene (500 mL) to a thick oil. 48% HBr (3 L) wasslowly and cautiously added to the reaction. Bubbling and foaming wasobserved during this addition which was exothermic. After the addition,the reaction became stirrable, and was stirred at reflux for 7 h. Thereaction was allowed to cool with stirring overnight. The reaction wasstirred with ice bath cooling and then suction filtered to collect anoff white solid. The solid was co-evaporated with toluene/methanol (1:1)and then dried under vacuum at 60° C. overnight. This afforded 197 g(77%) of (4) as an off white crystalline solid.

¹H NMR (300 MHz, CD₃OD) δ 1.66 (m, 4H), 2.57 (m, 2H), 2.92 (m, 2H), 6.70(d, J=8.5 Hz, 2H), 7.01 (d, J=8.5, Hz, 2H). CI MS m/z=166 [C₁₀H₁₅NO+H]⁺.

N-Cbz-4-(4-hydroxyphenyl)butylamine (29)

4-(4-Hydroxyphenyl)butylamine hydrobromide (4) (197 g, 0.80 mole), water(1 L), 1,4-dioxane (1 L) and sodium bicarbonate (336 g, 4 mole) werecombined and stirred while cooled in an ice-methanol cooling bath.Benzyl chloroformate (141 mL, 0.96 mole) was dripped in over 5 min. at−2° C. with no appreciable exotherm observed. This was stirred andallowed to warm to room temperature as the cooling bath thawedovernight. An additional quantity of benzyl chloroformate (8 mL, 0.54mol) was dripped in and this was allowed to stir for 2 h. The productmixture was then evaporated to approximately 500 mL and transferred to a2 L separatory funnel with ethyl acetate while decanting away from thesolids. The aqueous layer was extracted with ethyl acetate (3×1 L). Theextracts were combined, washed with brine, dried over sodium sulfate,filtered and evaporated to afford 265 g of the crude product. A portionof the crude product (130 g) was chromatographed (silica gel, 5:1hexanes/ethyl acetate) using toluene to load the column. The remainingcrude material was crystallized from 1:1 toluene/heptane. This materialwas suction filtered to collect the solid and washed with 1:1toluene/heptane.

This material was vacuum desiccated at 45° C. for 2 h. The combinedyield of compound (29) was 150 g (62%) of a white crystalline solid. ¹HNMR (300 MHz, CDCl₃) δ 1.43-1.65 (m, 4H), 2.52 (t, J=7.4 Hz, 2H), 3.19(q, J=6.4 Hz, 2H), 4.78 (br s, 1H), 5.09 (s, 2H), 5.77 (s, 1H), 6.74 (d,J=8.5 Hz, 2H), 6.98 (d, J=8.5 Hz, 2H), 7.34 (s, 5). CI MS m/z=300[C₁₈H₂₁NO₃+H]⁺.

N-Cbz-4-[4-(2,3-dihydroxypropyloxy)phenyl]butylamine (31)

N-Cbz-4-(4-hydroxyphenyl)butylamine (31) (30 g, 0.10 mole), glycidol(8.0 mL, 0.12 mole) ethanol (30 mL) and triethylamine (0.7 mL, 0.005mole) were stirred at reflux under argon for 2 h. The product mixturewas evaporated, taken up in hot ethyl acetate and suction filteredthrough a plug of silica gel, eluting with ethyl acetate. Afterevaporating to a white solid, this solid was re-crystallized fromtoluene to afford 21.8 g (58%) of compound (31).

¹H NMR (300 MHz, CD₃OD) δ 1.42-1.65 (m, 4H), 2.54 (t, J=7.5 Hz, 2H),3.11 (t, J=6.4 Hz, 2H), 3.58-3.71 (m, 2H), 3.88-4.04 (m, 3H), 5.05 (s,2H), 6.84 (d, J=8.7 Hz, 2H), 7.06 (d, J=8.5 Hz, 2H), 7.32 (s, 5H).

4-[4-(2,3-propanediol-1-oxy)phenyl]butylamine (32)

N-Cbz-4-[4-(2,3-dihydroxypropyloxy)phenyl]butylamine (31) (67 g, 0.179mole), ethanol (900 mL), acetic acid (50 mL) and 50% wet 10% palladiumon carbon (10 g) were stirred at atmospheric pressure under H₂. Afterstirring overnight, the reaction was purged with nitrogen and suctionfiltered through a pad of celite. This was evaporated and thenco-evaporated 3 times with ethanol (500 mL). The residue waschromatographed (silica gel, 100:10:1 methylenechloride/methanol/concentrated ammonium hydroxide) to afford 38 g (89%)of pure compound (32).

¹H NMR (300 MHz, CD₃OD) δ 1.42-1.55 (m, 2H), 1.55-1.68 (m, 2H), 2.56 (t,J=7.5 Hz, 2H), 2.65 (t, J=7.2 Hz, 2H), 3.58-3.72 (m, 2H), 3.89-4.05 (m,3H), 6.85 (d, J=8.7 Hz, 2H), 7.08 (d, J=8.7 Hz, 2H).

Example 54-[4-(2,3-Diacetoxypropyloxy)phenyl]butylamidino-3,5-diamino-6-chloropyrazinecarboxamide(36)

N-Cbz-4-[(2,3-diacetoxypropyloxy)phenyl]butylamine (34)

Acetic anhydride (0.6 ml, 6 mmol) was added to solution of 31 (0.55 g,1.5 mmol) in dry pyridine (50 mL) under stirring. The reaction mixturewas stirred 3 h at 25° C. and 3 h at 40° C. (oil bath). After this time,the reaction was quenched with 2N HCl (100 mL) and extracted with ethylacetate. The organic fraction was washed with water and dried oversodium sulfate. The solvent was removed under reduced pressure toprovide 34 (0.6 g 86%) as a white powder. ¹H NMR (300 MHz, CDCl₃) δ 1.55(m, 4H), 1.98 (s, 3H), 2.02 (s, 3H), 2.55 (m, 2H), 4.08 (m, 2H), 4.30(m, 2H), 4.45 (m, 1H), 4.80 (br s, 1H), 5.08 (s, 2H), 5.38 (m, 1H), 6.82(d, 2H), 7.08 (d, 2H), 7.35 (s, 5H).

4-[(2,3-diacetoxypropyloxy)phenyl]butylamine (35)

Cbz-protected amine 34 (0.6 g, 1.3 mmol) was dissolved in methanol (25mL) containing 1% acetic acid then Pd/C (0.22 g, 5% wet.) was added. Thereaction mixture was stirred for 3 h under hydrogen (1 atm), then thesolution was filtered through a pad of silica gel and the solvent wasevaporated to give amine 35 (0.37 g, 86%) as a clear oil.

4-[4-(2,3-Diacetoxypropyloxy)phenyl]butylamidino-3,5-diamino-6-chloropyrazinecarboxamide(36)

1-(3,5-Diamino-6-chloropyrazinoyl-2-methyl-pseudothiourea hydroiodide(0.37 g, 0.95 mmol) was added to a solution of 35 (0.27 g, 0.7 mmol) ina mixture of THF (40 mL) and diisopropylethylamine (1 mL). The reactionmixture was stirred at reflux (66° C.) for 6 h. After this time, thesolvent was evaporated and the residue was dissolved in MeOH. Silica gel(25 mL) was added and the solvent was removed under reduced pressure toadsorb the compound onto the silica gel. This silica gel was added tothe top of a silica gel column and flash chromatography (silica gel,10:1:0.1 chloroform/methanol/concentrated ammonium hydroxide) wasperformed to obtain crude 36 (128 mg). A second chromatography gave pure36 (14 mg, 3.7%) as a yellow powder. ¹H NMR (300 MHz, DMSO-d₆) δ 1.56(m, 4H), 2.05 (s, 6H), 2.58 (m, 2H), 3.14 (m, 2H), 4.10 (m, 2H), 4.28(m, 2H), 5.28 (br s, 1H), 6.58 (br s, 2H), 6.82 (m, 2H), 7.10 (m, 2H).APCI MS m/z=536[C₂₃H₃₀ClN₇O₆+H]⁺.

Example 64-[4-(2,2Dimethyl-[1,3]dioxolan-4yl)methyloxyphenyl]butylamidino-3,5-diamino-6-chloropyrazinecarboxamide(37)

4-[4-(2,2Dimethyl-[1,3]dioxolan-4yl)methyloxyphenyl]butylamidino-3,5-diamino-6-chloropyrazinecarboxamide(37)

Compound 33 (150 mg, 0.3 mmol) was suspended in dry acetone (50 mL) thenmethanol was added until a clear solution was formed (approx. 15 mL).p-Toluenesulfonic acid monohydrate (25 mg) was added along withmolecular sieves (5 A) and the reaction mixture was stirred for 48 h atroom temperature. After this time, the reaction mixture was filtered,silica gel (20 mL) was added and the solvent was removed under reducedpressure. This silica gel with the reaction mixture adsorbed was addedto the top of a silica gel flash chromatography column. Compound 37 (120mg, 81%) was isolated by flash chromatography (silica gel, 10:1:0.1chloroform/methanol/concentrated ammonium hydroxide) as a yellow solid.¹H NMR (300 MHz, DMSO-d₆) δ 1.30 (s, 3H), 1.34 (s, 3H), 1.52 (br s, 4H),2.56 (br s, 2H), 3.13 (br s, 2H), 3.71 (m, 1H), 3.92 (m, 2H), 4.08 (m,1H), 4.45 (m, 1H), 6.64 (br s, 2H), 6.82 (m, 2H), 7.12 (m, 2H). APCI MSm/z=492 [C₂₂H₃₀ClN₇O₄+H]⁺.

Example 74-[4-(Methyl-2,3,4-tri-O-acetyl-glucopyranonuronate-1-O-yl)phenyl]butylamidino-3,5-diamino-6-chloropyrazinecarboxamide(40)

2,3,4-Tri-O-acetyl-1-O-[4-(4-benzyloxycarbonylaminobutyl)phenyl]glucopyranuronicacid methyl ester (38)

2,3,4-Tri-O-acetyl-α-D-glucuronic acid methyl ester trichloroimidate(1.6 g, 3.3 mmol) was added under argon to protected aminophenol 29 indry methylene chloride (40 mL) then the solution was cooled to −25° C.After stirring for 10 min, BF₃.OEt₂ (0.045 mL, 0.33 mmol) was added inmethylene chloride (5 mL). The reaction mixture was stirred 1.5 h at−25° C., then allowed to warm up to −10° C. and stirring was continued 1h at that temperature. After this time, the temperature was increased to25° C. and the reaction mixture was stirred for 1 h then quenched withsaturated ammonium chloride (25 mL). The mixture was extracted withmethylene chloride then the organic fraction was washed with water anddried over sodium sulfate. The solvent was evaporated and the residuewas purified by flash chromatography (silica gel, 1:2 ethylacetate/hexanes) to provide 38 (1.5 g, 72%) as a white solid. ¹H NMR(300 MHz, DMSO-d₆) δ 1.41 (m, 2H), 1.51 (m, 2H), 1.99-2.02 (m, 9H), 2.54(m, 2H), 3.00 (m, 2H), 3.63 (s, 3H), 4.69 (m, 1H), 4.99 (s, 2H),5.04-5.10 (m, 2H), 5.46 (m, 1H), 5.60 (m, 1H), 6.88 (d, 2H), 7.12 (d,2H), 7.27 (m, 1H), 7.33 (s, 5H). APCI MS m/z=616 [C₃₁H₃₇NO₁₂+H]⁺.

2,3,4-Tri-O-acetyl-1-O-[4-(4-aminobutyl)phenyl]glucopyranuronic acidmethyl ester (39)

Glucuronide 38 (1.5 g, 2.4 mmol) was dissolved in dry methanol (100 mL)and Pd/C (0.62 g, 5%) was added. The reaction mixture was stirred underhydrogen (1 atm) for 2.5 h at room temperature. After this time, thesolution was passed through a pad of silica gel and the solvent wasevaporated under reduced pressure to give amine 39 (0.94 g, 84%). ¹H NMR(300 MHz, CDCl₃) δ 1.46 (m, 2H), 1.60 (m, 2H), 2.08 (m, 9H), 2.58 (m,2H), 2.72 (m, 2H), 3.63 (s, 3H), 4.14 (m, 1H), 5.10 (m, 1H), 5.34 (m,3H), 6.90 (d, 2H), 7.12 (d, 2H).

4-[4-(Methyl2,3,4-tri-O-acetyl-glucopyranonuronate-1-O-yl)phenyl]butylamidino-3,5-diamino-6-chloropyrazinecarboxamide(40)

1-(3,5-Diamino-6-chloropyrazinoyl-2-methyl-pseudothiourea hydroiodide(0.30 g, 0.8 mmol) was added to a solution of 39 (0.4 g, 0.8 mmol) in amixture of THF (40 mL) and diisopropylethylamine (3 mL). The reactionmixture was stirred at reflux (66° C.) for 2 h. After this time, thesolvent was evaporated and the residue was suspended in THF. Silica gel(15 mL) was added and the solvent was removed under reduced pressure.This silica gel was transferred onto the top of a silica gelchromatography column. The target compound 40 (0.32 g, 48%) was purifiedby flash chromatography (silica gel, 12:1:0.1chloroform/ethanol/concentrated ammonium hydroxide) and isolated as ayellow powder. ¹H NMR (300 MHz, CDCl₃) δ 1.61 (br s, 4H), 2.05 (s, 9H),2.55 (m, 2H), 3.49 (br s, 2H), 3.71 (m, 3H), 4.22 (m, 1H), 5.12 (m, 1H),5.34 (m, 3H), 6.88 (m, 2H), 7.04 (d, 2H). APCI MS m/z=694[C₂₉H₃₆ClN₇O₁₁+H]⁺.

Example 84-[4-(5-Carboxy-glucopyranonuronate-1-O-yl)-phenyl]butylamidino-3,5-diamino-6-chloropyrazinecarboxamide(41)

4-[4-(5-Carboxy-2,3,4-tri-O-acetyl-glucopyranonuronate-1-O-yl)phenyl]butylamidino-3,5-diamino-6-chloropyrazinecarboxamide(41)

Compound 40 (0.31 g, 0.44 mmol) was added to mixture of THF/water (1:1,40 mL) and the resulting cloudy solution was cooled to −10° C. Asolution of NaOH in water (4 mL of 1.24 N solution) was added andstirring was continued at 10° C. for 1.5 h. After this time, thereaction mixture was allowed to warm up to room temperature and the THFwas removed under reduced pressure. The pH of the remaining solution wasadjusted to 6 by drop wise addition of 1N HCl. The formed precipitatewas collected by centrifugation and washed with cold water (3×20 mL).Compound 41 (0.18 g, 75%) was isolated as a yellow powder after dryingunder vacuum for 48 h. ¹H NMR (300 MHz, DMSO-d₆) δ 1.56 (br s, 4H), 2.56(m, 2H), 3.19 (m, 2H), 3.15-3.40 (br s., 3.25 (m, 2H), 3.57 (m, 1H),4.87 (m, 1H), 5.10-5.40 (br d, 1H), 6.89 (m, 2H), 7.06 (m, 2H). APCI MSm/z=554 [C₂₉H₃₆ClN₇O₁₁+H]⁺.

Example 94-[4-(5-Carboxy-glucopyranonuronate-1-O-yl)phenyl]butylamidino-3,5-diamino-6-chloropyrazinecarboxamidetrifluoroacetate (42)

4-Methylphenylsulfonic acid 4-(4-methoxyphenyl)butyl ester (1)

Pyridine (15 mL) was added drop wise to a cooled (0° C.) solution of4-(4-methoxyphenyl)butanol (10.0 g, 0.055 mol) and p-toluenesulfonylchloride (13.6 g, 0.072 mol) in dry chloroform (100 mL) under stirring.The reaction mixture was stirred overnight at room temperature. Afterthis time, the reaction was quenched with 10% HCl (300 mL) and extractedwith chloroform. The organic fraction was washed with saturated NaHCO₃,water and dried over magnesium sulfate. The solvent was removed underreduced pressure and the residue purified by flash chromatography(eluent: hexane/ethyl acetate 15:1) to provide 12.9 g (66%) of 1 asclear oil. ¹H NMR (360 MHz, CDCl₃) δ 1.61 (m, 4H), 2.44 (s, 3H), 2.52(m, 2H), 3.78 (s, 3H), 4.05 (m, 2H), 6.77 (d, 2H), 7.05 (d, 2H), 7.34(d, 2H), 7.78 (d, 2H).

4-(4-Methoxyphenyl)butylazide (2)

Sodium azide (3.07 g, 0.047 mol) was added to a solution of 1 (12.9 g,0.04 mol) in anhydrous DMF (70 mL) and the reaction mixture was stirred12 h at 80° C. (oil bath). Then solvent was removed at reduced pressureand the residual oil was treated with a mixture of CH₂Cl₂/ether 3:1 (100mL). The resulting solution was washed with water (2×100 mL), brine anddried over magnesium sulfate. The solvent was removed under reducedpressure and 7.6 g (95%) of 2 was obtained. The purity of 2 (99%) wasdetermined by GC and TLC (eluent: hexane/ethyl acetate 1:1), R_(f)=0.84.

4-(4-Methoxyphenyl)butylamine (3). Typical Procedure A

Lithium aluminum hydride (LAB) (55 mL of a 1.0 M solution in THF, 0.055mol) was added drop wise to a solution of 2 (7.6 g, 0.037 mol) in dryTHF (70 mL) at 0° C. The mixture was stirred overnight at roomtemperature in an argon atmosphere then the mixture was treated withwater (1.5 mL), then 15% NaOH (1.5 mL), then with more water (3 mL) andfiltered. The solid precipitate was washed with THF. The combinedorganic fractions were dried over magnesium sulfate and the solvent wasremoved under reduced pressure to give 6.2 g (94%) of 3. The purity of 3(99%) was determined by GC. ¹H NMR (360 MHz, DMSO-d₆) δ 1.34 (m, 2H),1.54 (m, 2H), 2.51 (m, 4H), 3.70 (s, 3H), 6.83 (d, 2H), 7.08 (d, 2H).¹³C (90 MHz, DMSO-d₆) δ 28.6, 330, 34.1, 41.5, 54.8, 113.1, 129.1,132.2, 157.3

4-(4-Hydroxyphenyl)butylamine hydrobromide (4). Typical Procedure B

Amine 3 (2.32 g, 0.012 mol) was stirred in boiling 48% HBr (50 mL) for 3h. After the reaction was completed, argon was bubbled through thesolution and the solvent was evaporated under reduced pressure. Thesolid residue was dried above KOH to provide 3.1 g (90%) of 4. APCI MSm/z=166[C₁₀H₁₅NO+H]⁺

4-(4-Hydroxyphenyl)butylamidino-3,5-diamino-6-chloropyrazinecarboxamidehydrochloride (5)

1-(3,5-Diamino-6-chloropyrazinoyl-2-methyl-pseudothiourea hydroiodide(0.4 g, 1.03 mmol) was added to a suspension of4-(4-hydroxyphenyl)butylamine hydrobromide (4) (0.8 g, 32 mmol) in amixture of THF (35 mL) and triethylamine (3 mL). The reaction mixturewas stirred in the boiling solvent for 3 h, then the supernatant wasseparated and the solvent was removed under reduced pressure. The oilyresidue was washed with water (2×30 mL), ether (3×30 mL) and then 10%HCl (40 mL) was added. The mixture was vigorously stirred for 10 minthen the yellow solid was filtered off, dried and recrystallized twicefrom ethanol to give 5 (0.18 g, 41%) as yellow solid. Purity is 98% byHPLC, retention time is 9.77 min. ¹H NMR (300 MHz, DMSO-d₆) δ 1.56 (brs, 4H), 2.48 (br s, 2H), 3.35 (m, 2H), 6.65 (d, 2H), 6.95 (d, 2H), 7.50(br s, 2H), 8.75 (br s, 1H), 9.05 (br s, 1H), 9.33 (br s, 2H), 10.55 (s,1H). ¹³C NMR (75 MHz, CD₃OD) 28.7, 29.8, 35.4, 42.4, 111.2, 116.1,122.0, 130.0, 134.0, 155.0, 156.1, 156.8, 157.5, 167.0. APCI MS m/z=378[C₁₆H₂₀ClN₇O₂+H]⁺.

4-[4-(5-Carboxy-glucopyranonuronate-1-O-yl)phenyl]butylamidino-3,5-diamino-6-chloropyrazinecarboxamidetrifluoroacetate (42)

Compound 5 (29 mg, 0.07 mmol) was dissolved in DMF (2 mL). A 100 mM TRISbuffer solution, pH 7.5, containing 10 mM MgCl₂, 1.0 mM dithiothreitol,10 mM saccharolactone, and 2 mM CMP (cytidine-mono-phosphate) was made.176 mg of UDP-GA (uridine-di-phospho-glucuronic acid) was dissolved inthe buffer solution (30 mL) and added to 600 mg of bovine livermicrosomes (produced at AMRI Biocatalysis Division) in a 50 mL widemouthjar. The DMF solution of 5 was added to initiate the reaction. Thereaction was run at room temperature with periodic shaking by hand andwas stopped by the addition of an equal volume of MeCN after 42 h. Thereaction solution was divided into two (50 mL) centrifuge tubes andcentrifuged to remove the enzyme. The precipitated enzyme wasre-suspended in MeCN (40 mL) and centrifuged again. This enzyme wash wasrepeated 3 times until the LC/MS analysis showed only trace amounts ofremaining product. The supernatants were combined and the aqueous MeCNwas removed under vacuum at 30° C. The resulting syrup was thinned bythe addition of MeCN and further dried under vacuum overnight. Afterdrying, the syrup was purified by RP-HPLC (Luna C18 (2) 250×21.2 mm, 5μm) with a water/acetonitrile (both containing 0.1% TFA) gradient. Theappropriate fractions were combined and dried under vacuum to yield 42(14.8 mg, 28.2%) with 98.4% purity (by HPLC analysis) as a white solid.APCI MS m/z=554 [C₂₉H₃₆ClN₇O₁₁+H]⁺, m/z=552[C₂₉H₃₆ClN₇O₁₁−H]⁻.

Example 104-[4-(1,4-Dioxapent-1-yl)phenyl]butylamidino-3,5-diamino-6-chloropyrazinecarboxamidehydrochloride (66)

N-Cbz-4-[4-(1,4-dioxapent-1-yl)phenyl]butylamine (64)

To a vigorously stirred solution of 29 (4 g, 0.013 mol) in anhydrous THF(150 mL) under nitrogen at 0° C. was added sodium hydride (60%dispersion in mineral oil, 0.64 g, 0.016 mol). The mixture was stirredfor 15 min then tetrabutylammonium iodide (0.5 g, 0.0013 mol) and2-bromoethyl methyl ether (2.04 g, 0.015 mol) were added and the mixturewas stirred at room temperature overnight. The solvent was removed underreduced pressure and the material was purified by column chromatography(silica gel, 10:1 methylene chloride/ethyl acetate) to provide 64 (3.1g, 64%). ¹H NMR (300 MHz, CDCl₃) δ 1.60 (m, 4H), 2.59 (m, 2H), 3.23 (m,2H), 3.45 (s, 3H), 3.77 (m, 2H), 4.10 (m, 2H), 5.13 (s, 2H), 6.88 (d,2H), 7.08 (d, 2H), 7.47 (s, 5H).

4-(1,4-Dioxapent-1-yl)phenylbutylamine (65)

To a solution of 64 (2.27 g, 6.4 mmol) in ethanol (60 mL) with aceticacid (1 wt. %) was added Pd/C catalyst (300 mg, 10% wet) then themixture was shaken for 18 h at 30 psi of hydrogen in a Parrhydrogenator. The pressure was released and the catalyst was filteredoff through a pad of silica gel. The solvent was removed at reducedpressure to provide 65 (1.3 g, 92%). ¹H NMR (300 MHz, CDCl₃) δ 1.60 (brs, 4H), 2.00 (s, 2H) 2.55 (br s, 2H), 2.85 (br s, 2H), 3.47 (s, 3H),3.73 (m, 2H), 4.10 (m, 2H), 6.82 (d, 2H), 7.08 (d, 2H).

4-[4-(1,4-Dioxapent-1-yl)phenyl]butylamidino-3,5-diamino-6-chloropyrazinecarboxamidehydrochloride (66)

1-(3,5-Diamino-6-chloropyrazinoyl-2-methyl-pseudothiourea hydroiodide(0.3 g, 0.77 mmol) was added to an anhydrous THF solution (20 mL) of 65(0.48 g, 2.3 mmol). The reaction mixture was stirred at reflux for 11 hthen the solvent was evaporated. The residue was purified on a Biotagesystem (silica gel, 10:1:0.1 chloroform/methanol/concentrated ammoniumhydroxide). The appropriate fractions were collected, the solvent wasevaporated and the residue was treated with 3% HCl. The yellowprecipitate that formed was separated and washed with ethyl acetate,water and dried to provide 66 (160 mg, 34%) as a yellow solid. ¹H NMR(300 MHz, DMSO-d₆) δ 1.57 (br s, 4H), 2.52 (m, 4H), 3.30 (s, 3H), 3.63(m, 2H), 4.03 (m, 2H) 6.85 (d, 2H), 7.12 (d, 2H), 7.46 (br s, 2H), 8.00(br s, 1H), 8.85 (br s, 1H), 8.99 (br s, 1H), 9.32 (br s, 1H), 10.03 (s,1H). APCI MS m/z 436 [C₁₉H₂₆ClN₇O₃+H]⁺.

Example 114-(4-Hydroxymethylphenyl)butylamidino-3,5-diamino-6-chloropyrazinecarboxamidehydrochloride (68)

4-(4-Hydroxymethylphenyl)butyl amine (67)

Lithium aluminum hydride (35 mL of a 1.0 M solution in THF, 0.035 mol)was added drop wise to a vigorously stirred solution of4-(4-carboxymethylphenyl)butylazide 8 (2.4 g, 0.010 mol) in dry THF (120mL) at 0° C. and stirred overnight at room temperature under a nitrogenatmosphere. To break up the complex water (1.5 mL), 15% NaOH (1.5 mL)and water (4.5 mL) were added drop wise to the cold reaction mixture.The white solid precipitate was filtered off and washed with THF. Allorganics phases were combined and evaporated. The material was purifiedby column chromatography (silica gel, 2:1:0.05chloroform/ethanol/concentrated ammonium hydroxide) to provide 67 (1.17g, 64%) as a white solid. ¹H NMR (300 MHz, CDCl₃) δ 1.15 (br s, 2H),1.54 (br s, 2H) 1.70 (br s, 2H), 2.60 (m, 4H), 3.77 (s, 1H), 4.67 (s,2H), 7.47 (s, 2H), 7.60 (s, 2H).

4-(4-Hydroxymethylphenyl)butylamidino-3,5-diamino-6-chloropyrazinecarboxamidehydrochloride (68)

1-(3,5-Diamino-6-chloropyrazinoyl-2-methyl-pseudothiourea hydroiodide(0.2 g, 0.52 mmol) was added to an anhydrous THF suspension (20 mL) of67 (0.37 g, 2.06 mmol). The reaction mixture was stirred at reflux for 3h then the solvent was evaporated. The residue was washed with ethylacetate (3×15 mL), dried and treated with 3% HCl (15 mL). The yellowsolid that formed was filtered, washed with water (2×10 mL) and dried toprovide 68 (216 mg, 98%). ¹H NMR (300 MHz, DMSO-d₆) δ 1.57 (br s, 4H),2.62 (m, 2H), 3.35 (m, 2H), 3.73 (br s, 4H), 4.45 (s, 2H), 7.12 (d, 2H),7.24 (d, 2H), 8.85 (br s, 1H), 9.98 (br s, 1H), 9.32 (br s, 1H), 10.55(s, 1H). APCI MS m/z 392 [C₁₇H₂₂ClN₇O₃+H]⁺.

Example 124-{4-[(2R)-2,3-Dihydroxypropyloxy]phenyl]}butylamidino-3,5-diamino-6-chloropyrazinecarboxamidehydrochloride (71)

N-Cbz-4-{[(2R)-2,3-dihydroxypropyloxy]phenyl}butylamine (69)

To cold (0° C.) N-Cbz-4-(4-allyloxyphenyl)butylamine 30 (1.94 g, 5.7mmol) under a nitrogen atmosphere was added cold (0° C.) AD-Mix-α (12 g)in tert-butanol (100 mL) and water (100 mL). The mixture was allowed towarm to room temperature overnight. The mixture was then quenched withsaturated sodium sulfite (200 mL), extracted with ethyl acetate (3×100mL), dried (Na₂SO₄) and concentrated to give 69 (2 g, 95%) as a beigesolid. ¹H NMR (300 MHz, DMSO) δ 1.40 (m, 2H), 1.54 (m, 2H) 2.55 (br s,2H), 3.00 (m, 2H), 3.44 (s, 2H), 3.78 (m, 2H), 3.94 (m, 1H), 4.68 (br s,1H), 4.93 (s, 1H), 5.00 (s, 2H), 6.83 (d, 2H), 7.08 (d, 2H), 7.30 (br s,1H), 7.35 (s, 5H).

4-{[(2R)-2,3-dihydroxypropyloxy]phenyl}butylamine (70)

To a vigorously stirred solution of 69 (2 g, 5.4 mmol) in methanol (60mL) under nitrogen was added Pd/C (10% wet, 0.5 g). The mixture wasstirred 2 h at room temperature under an atmosphere of hydrogen then thepressure was released and the mixture was filtered through a pad ofsilica gel. The solvent was removed and the residue was purified bycolumn chromatography (silica gel, 2:1:0.2chloroform/ethanol/concentrated ammonium hydroxide) to provide 70 (1.18g, 92%) as a white solid. ¹H NMR (300 MHz, DMSO-d₆) δ 1.32 (m, 2H), 1.54(m, 2H), 2.55 (m, 2H), 3.45 (m, 2H), 3.82 (m, 3H), 3.94 (m, 2H), 6.83(d, 2H), 7.08 (d, 2H).

4-{4-[(2R)-2,3-Dihydroxypropyloxy]phenyl]}butylamidino-3,5-diamino-6-chloropyrazinecarboxamidehydrochloride (71)

1-(3,5-Diamino-6-chloropyrazinoyl-2-methyl-pseudothiourea hydroiodide(0.4 g, 1.03 mmol) was added to an anhydrous THF suspension (50 mL) of70 (0.49 g, 2.00 mmol). The reaction mixture was stirred at reflux for 5h then the mixture was cooled and the precipitate that formed wascollected and washed with 3% HCl (2×5 mL) then dried to provide 71 (290mg, 58%) as a yellow solid. ¹H NMR (300 MHz, DMSO-d₆) δ 1.57 (s, 4H),2.55 (d, 2H), 3.35 (d, 2H), 3.90 (m, 5H), 6.82 (d, 2H), 7.10 (d, 2H),7.47 (br s, 2H), 8.75 (br s, 1H), 8.90 (br s, 1H), 10.5 (s, 1H). APCI MSm/z 452 [C₁₉H₂₆ClN₇O₄+H]⁺.

Example 134-{4-[(2S)-2,3-Dihydroxypropyloxyl]phenyl]}butylamidino-3,5-diamino-6-chloropyrazinecarboxamidehydrochloride (74)

N-Cbz-4-{[(2S)-2,3-dihydroxypropyloxy]phenyl}butylamine (72)

To cold (0° C.) N-Cbz-4-(4-allyloxyphenyl)butylamine 30 (1.53 g, 4.5mmol) under a nitrogen atmosphere was added cold (0° C.) AD-Mix-β (9.2g) in tert-butanol (100 mL) and water (100 mL). The mixture was allowedto warm to room temperature overnight. The mixture was quenched withsaturated sodium sulfite (200 mL), extracted with ethyl acetate (3×100mL), dried (Na₂SO₄) and concentrated to provide 72 (1.67 g, 99%) as abeige solid. ¹H NMR (300 MHz, CDCl₃) δ 1.54 (m, 4H) 2.55 (br s, 2H),3.18 (m, 2H), 3.70 (s, 3H), 4.02 (d, 2H), 4.10 (m, 1H), 4.73 (br s, 1H),5.08 (s, 2H), 6.83 (d, 2H), 7.08 (d, 2H), 7.38 (s, 1H), 7.35 (s, 5H).

4-{[(2S)-2,3-dihydroxypropyloxy]phenyl}butylamine (73)

Compound 73 was prepared in a similar fashion to the synthesis ofcompound 70 starting from compound 72 (1.67 g, 4.5 mmol). Amine 73 (1.06g, 99%) was isolated as a white solid. ¹H NMR (300 MHz, DMSO-d₆) δ 1.32(m, 2H), 1.54 (m, 2H), 2.55 (m, 2H), 3.45 (m, 2H), 3.75 (m, 3H), 3.94(m, 2H), 6.83 (d, 2H), 7.08 (d, 2H).

4-{4-[(2S)-2,3-Dihydroxypropyloxy]phenyl]}butylamidino-3,5-diamino-6-chloropyrazinecarboxamidehydrochloride (74)

Compound 74 was prepared in a similar fashion to the synthesis ofcompound 71 starting from compound 73 (0.74 g, 3.09 mmol). Compound 74(0.38 g, 76%) was isolated as a yellow solid. ¹H NMR (300 MHz, DMSO-d₆)δ 1.57 (s, 4H), 2.55 (d, 2H), 3.35 (d, 2H), 3.85 (m, 5H), 6.82 (d, 2H),7.10 (d, 2H), 7.47 (br s, 2H), 8.75 (br s, 1H), 8.90 (br s, 1H), 10.5(s, 1H). APCI MS m/z 452 [C₁₉H₂₆ClN₇O₄+H]⁺.

Example 144-(4-Aminophenyl)ethylamidino-3,5-diamino-6-chloropyrazinecarboxamidehydrochloride (83)

4-(4-Aminophenyl)ethylamidino-3,5-diamino-6-chloropyrazinecarboxamidehydrochloride (83)

A mixture of 1-H-pyrazolecarboxamidine hydrochloride (2.8 g, 19 mmol),4-aminoethylaniline (1.3 mL, 9 mmol) and diisopropylethylamine (1.3 ml)were stirred in dry DMF (5 mL) under argon for 18 h. After this time,ether (30 ml) was added to produce a clear oil. The obtained oil (82)was washed with ether and dried under vacuum (40 mTorr) overnight. Afterdrying 70 mg of oil was taken into dry methanol (3 mL) and 25% NaOH(0.14 mL) was added. The reaction volume was decreased to 1.0 mL and3,5-diamino-6-chloropyrazine-2 carboxylic acid methyl ester (0.1 g, 0.5mmol) was added. The mixture was stirred at room temperature overnight.Another portion of 82 (0.1 g) was dissolved in methanol (1 mL), treatedwith 25% NaOH (0.15 mL) and the resulting solution was added to thereaction mixture. The reaction mixture was stirred 3 h at reflux, thencooled to room temperature and the solvent was removed under reducedpressure. The residue was dissolved in a minimal volume of DMF andseparated by preparative HPLC. The obtained fractions were analyzed byLC/MS. The fractions containing product with M+H=349 were collected andthe solvent was removed under reduced pressure. The residue wasdissolved in 10% HCl and evaporated to dryness to produce 83 (23.5 mg,11%) as a yellow solid. ¹H NMR (360 MHz, DMSO-d₆) δ 2.91 (m, 2H), 3.59(m, 2H), 7.31 (d, 2H), 7.42 (m, 4H), 9.02 (br s., 2H), 9.41 (br s., 1H),10.56 (s, 1H). ¹³C NMR (90 MHz, DMSO-d₆) 33.1, 42.0, 108.9, 119.6,120.7, 129.9 (2C), 131.0 (2C), 153.1, 154.1, 155.8, 165.2. API MSm/z=349 [C₁₄H₁₇ClN₈O+H]⁺.

Preparative HPLC was performed on a Gilson Combichem using a Luna C18(2)column, 5/h, 250×21.2 mm; Flow rate=20 mL/min; Mobile phase consists ofMeCN/water containing 0.1% TFA; Gradient: 10% MeCN from the 0-2 mininterval, concentration of MeCN increased from 10 to 40% from 2-10 min,40 to 100% MeCN from 10-19 min, 100% MeCN from 19-23 min, MeCN decreasedfrom 100 to 10% from 23-25 min.

Example 154-[4-(1,4,7-Trioxaoct-1-yl)phenyl]-butylamidino-3,5-diamino-6-chloropyrazinecarboxamidehydrochloride (108)

4-[4-(1,4,7-Trioxaoct-1-yl)phenyl]-N-benzyloxycarbonylbutylamine (106)

4-(4-Hydroxyphenyl)-N-benzyloxycarbonylbutylamine (29) (1.0 g, 3.3mmol), 1-bromo-2-(2-methoxyethoxy)ethane (0.67 g, 3.7 mmol), andpotassium carbonate (0.60 g, 4.3 mmol) were combined in acetone (20 mL),and stirred at reflux overnight. The reaction was allowed to cool, andthen filtered and evaporated. The residue was re-subjected in methylethyl ketone (10 mL), 1-bromo-2-(2-methoxyethoxy)ethane (0.91 g, 5.0mmol), potassium carbonate (0.74 g, 5.3 mmol), and sodium iodide (0.5 g,3.3 mmol) with stirring at reflux for 2.5 h. The reaction was allowed tocool, and was filtered and evaporated. The residue was re-subjected inDMF (10 mL), with 1-bromo-2-(2-methoxyethoxy)ethane (1.8 g, 9.8 mmol),potassium carbonate (1.60 g, 11.6 mmol), and sodium iodide (0.4 g, 2.7mmol), overnight with stirring at 70° C. The reaction was evaporated toremove the solvent and then was dissolved in ethyl acetate (70 mL). Theorganic extract was washed with water (3×20 mL), dried over potassiumcarbonate and filtered. Evaporation afforded 1.1 g of an oil which waspurified by column chromatography (silica gel, 2:1 hexanes/ethylacetate) to afford 800 mg (70%) of pure product 106. ¹H NMR (300 MHz,CDCl₃) δ 1.42-1.68 (m, 4H), 2.55 (t, J=7.5 Hz, 2H), 3.20 (q, J=6.2 Hz,2H), 3.39 (s, 3H), 3.55-3.60 (m, 2H), 3.69-3.74 (m, 2H), 3.82-3.87 (m,2H), 4.11 (t, 5.3 Hz, 2H), 4.71 (br s, 1H), 5.09 (s, 2H), 6.82 (d, J=8.5Hz, 2H), 7.05 (d, J=8.5 Hz, 2H), 7.34 (s, 5H). CI MS m/z=402[C₂₃H₃₁NO₅+H]⁺.

4-[4-(1,4,7-Trioxaoct-1-yl)phenyl]butylamine (107)

Compound 106 (800 mg, 2.0 mmol) in ethanol (20 mL), was subjected to 1atmosphere of H₂ in the presence of 10% palladium on carbon (100 mg,cat.) with stirring for 5 h. After standing overnight, the reaction waspurged with N₂ then suction filtered through celite and washed off thecelite with methylene chloride. The solvents were evaporated andchromatographed (silica gel, 200:10:1 methylenechloride/methanol/concentrated ammonium hydroxide) to afford 530 mg(>99%) of amine 107. ¹H NMR (300 MHz, CDCl₃) δ 1.31 (br s, 2H),1.41-1.52 (m, 2H), 1.55-1.67 (m, 2H), 2.56 (t, J=7.7 Hz, 2H), 2.70 (t,J=7.0 Hz, 2H), 3.39 (s, 3H), 3.58 (m, 2H), 3.72 (m, 2H), 3.84 (t, J=4.9Hz, 2H), 4.12 (t, J=5.3 Hz, 2H), 6.83 (d, J=8.7 Hz, 2H), 7.07 (d, J=8.7Hz, 2H). CI MS m/z=268 [C₁₅H₂₅NO₃+H]⁺.

4-[4-(1,4,7-Trioxaoct-1-yl)phenyl]-butylamidino-3,5-diamino-6-chloropyrazinecarboxamidehydrochloride (108)

Amine 107 (200 mg, 0.75 mmol),1-(3,5-Diamino-6-chloropyrazinoyl-2-methyl-pseudothiourea hydroiodide(296 mg, 0.76 mmol) and triethylamine (0.52 mL, 3.73 mmol) were combinedin THF (5 mL) and stirred at reflux under N₂ for 1.5 h. After stirringat room temperature for two days, the reaction was evaporated. Theresidue was chromatographed (silica gel, 400:10:1 to 200:10:1 gradientelution, methylene chloride/methanol/concentrated ammonium hydroxide) toafford the free base of the product (290 mg). This material was stirredin methanol (20 mL) at 0° C. then 1M HCl (3 mL) was added. The solutionwas evaporated with no heating and then co-evaporated with methanol. Theresidue was dissolved in methanol and then precipitated by the additionof ethyl acetate. This precipitate was centrifuged and the supernatantwas decanted. The gel like pellet was evaporated, co-evaporated withwater (2 mL) and then placed on high vacuum overnight. This afforded 129mg (42%) of compound 108 as a yellow solid. ¹H NMR (300 MHz, DMSO-d₆) δ1.58 (m, 4H), 2.58 (br s, 2H), 3.25 (s, 3H), 3.33 (m, 2H), 3.45 (m, 2H),3.58 (m, 2H), 3.72 (m, 2H), 3.04 (m, 2H), 4.92 (br s, 4H), 6.85 (d,J=8.5 Hz, 2H), 7.12 (d, J=8.4 Hz, 2H), 8.10 to 7.26 (br m, 3H), 8.94 (brd, 2H), 9.32 (br s, 1H), M.P.=110-125 APCI MS m/z=480 [C₂₁H₃₀ClN₇O₄+H]⁺.

Example 164-[4-(1,4,7,10,13-pentoxatetradec-1-yl)phenyl]-butylamidino-3,5-diamino-6-chloropyrazinecarboxamidehydrochloride (112)

O-Tosyltetraethyleneglycol methyl ether (109)

Tetraethyleneglycol monomethyl ether (2.0 g, 9.6 mmol) was combined withpyridine (0.93 mL, 11.5 mmol) in methylene chloride (20 mL) at 0° C.p-Toluenesulfonyl chloride (2.2 g, 11.5 mmol) dissolved in methylenechloride (10 mL) was added dropwise and the reaction was allowed to warmto room temperature as the ice bath thawed. After stirring nine days,the product mixture was transferred to a separatory funnel with water(70 mL). The layers were separated and the aqueous layer was extractedwith methylene chloride (3×20 mL). The extracts were combined andevaporated. The residue (3.3 g) was chromatographed (silica gel, 4:1 to3:1 gradient elution, methylene chloride/ethyl acetate) to afford 2.5 g(70%) of compound 109 as an oil. ¹H NMR (300 MHz, DMSO-d₆) δ 1.56 (br s,4H), 2.42 (br s, 2H), 3.34 (br s, 4H), 6.05 (s, 3H), 7.09 (s, 0.5H),7.26 (s, 0.5H), 7.42 (br s, 2H), 7.70 (hr s, 2H), 8.87 (br d, 2H), 9.07(s, 2H), 9.22 (br s, 1H), 10.51 (s, 1H). CI MS m/z=363 [C₁₆H₂₆O₇S+H]⁺.

4-[4-(1,4,7,10,13-pentoxatetradec-1-yl)phenyl]-N-benzyloxycarbonylbutylamine(110)

4-(4-Hydroxyphenyl)-N-benzyloxycarbonylbutylamine (29) (0.30 g, 1.0mmol), O-tosyltetraethyleneglycol methyl ether (109) (1.45 g, 4.0 mmol),potassium carbonate (0.69 g, 5 mmol) and sodium iodide (0.6 g, 4.0 mmol)were combined in DMF (5 mL) and stirred at 55° C. overnight. Cesiumcarbonate (0.33 g, 1.0 mmol) was added and the reaction was stirred at70° C. overnight. The mixture was allowed to cool and was thenpartitioned between 1:1 toluene/ethyl acetate (70 mL) and water (20 mL).The layers were separated and the organic layer was washed with water(4×10 mL), brine (2×30 mL), dried over sodium sulfate and evaporated.Chromatography (silica gel, 3:1 methylene chloride/ethyl acetate)afforded 400 mg (81%) of compound 110. ¹H NMR (300 MHz, CDCl₃) δ1.44-1.67 (m, 4H), 2.55 (t, J=7.7 Hz, 2H), 3.20 (q, J=6.0 Hz, 2H), 3.37(s, 3H), 3.54 (m, 2H), 3.61-3.75 (m, 10H), 3.84 (t, J=4.9 Hz, 2H), 4.10(t, 5.5 Hz, 2H), 4.71 (br. s, 1H), 5.09 (s, 2H), 6.82 (d, J=8.5 Hz, 2H),7.05 (d, J=8.6 Hz, 2H), 7.34 (s, 5H).

4-[4-(1,4,7,10,13-pentoxatetradec-1-yl)phenyl]butylamine (111)

This compound was prepared in a similar fashion to the synthesis ofamine 107 from compound 110 (385 mg, 0.78 mmol) to give 266 mg (95%) ofcompound 111. ¹H NMR (300 MHz, CDCl₃) δ 1.41-1.54 (m, 2H), 1.60 (br. s,2H), 2.56 (t, J=7.5 Hz, 2H), 2.70 (t, J=7.0 Hz, 2H), 3.37 (s, 3H),3.51-3.58 (m, 2H), 3.60-3.77 (m, 10H), 3.84 (m, 2H), 4.10 (t, 5.5 Hz,2H), 6.83 (d, J=8.3 Hz, 2H), 7.07 (d, J=8.7 Hz, 2H).

4-[4-(1,4,7,10,13-pentoxatetradec-1-yl)phenyl]butylamidino-3,5-diamino-6-chloropyrazinecarboxamidehydrochloride (112)

This compound was prepared in a similar fashion to the synthesis ofcompound 108 from compound 111 (250 mg, 0.70 mmol) and1-(3,5-diamino-6-chloropyrazinoyl-2-methyl-pseudothiourea hydroiodide(200 mg, 0.51 mmol) to give 130 mg (46%) of compound 112. ¹H NMR (300MHz, DMSO-d₆) δ 1.57 (br s, 4H), 2.55 (br s, 2H), 3.05 to 3.90 (m, 21H),6.85 (d, J=7.6 Hz, 2H), 7.12 (d, J=7.4 Hz, 2H), 7.44 (br s, 1H),8.10-7.40 (m, 2H), 8.93 (br d, 2H), 9.32 (s, 1H), 10.55 (s, 1H). APCI MSm/z=568 [C₂₅H₃₈ClN₇O₆S+H]⁺.

Example 174-[4-(2-Hydroxyethyloxy)phenyl)]butylamidino-3,5-diamino-6-chloropyrazinecarboxamidehydrochloride (116)

N-{4-[4-(2-hydroxyethyloxy)phenyl]but-3-yn-1-yl}phthalimide (113)

2-(4-Bromophenoxy)ethanol (3 g, 14.5 mmol), palladium (II) chloride (0.2g, 1.1 mmol) and triphenylphosphine (0.6 g, 2.2 mmol) were dissolved intriethylamine (70 mL) then copper(I) iodide (0.45 g, 2.1 mmol) andN-(but-3-yn)phthalimide (3.2 g, 16 mmol) were added. The reactionmixture was stirred for 72 h at room temperature and 5 h at 50° C. (oilbath), then the solvent was removed under reduced pressure. Ethylacetate (150 mL) was added to the residue and the mixture was washedwith 2N HCl, brine and water. The organic fraction was isolated, driedwith sodium sulfate and the solvent was removed under reduced pressure.The product 113 (1.7 g, 43%) was isolated by flash chromatography(silica gel, 10:1:2 methylene chloride/ethyl acetate/hexanes) as a brownoil. ¹H NMR (300 MHz, CDCl₃) δ 2.80 (m, 2H), 3.95 (m, 4H), 4.08 (m, 2H),6.83 (d, 2H), 7.35 (m, 2H), 7.72 (m, 2H), 7.88 (m, 2H).

N-{4-[4-(2-hydroxyethyloxy)phenyl]butyl}phthalimide (114)

A solution of 113 (1.7 g, 5.1 mmol) in a mixture of methanol/ethylacetate (80 and 10 mL correspondingly) was placed in a 0.5 L Parr flaskand palladium on carbon (1.1 g, 5% wet. Pd/C) was added. The reactionmixture was shaken at 50 psi of hydrogen pressure at room temperatureovernight. After this time, the mixture was filtered through a silicagel pad and the solvent was removed at reduced pressure to give crude114 (1.2 g) as a brown oil. The crude 114 was used in the next stepwithout further purification.

4-[4-(2-hydroxyethyloxy)phenyl]butyl amine (115)

The crude protected amine 114 (1.2 g, 35 mmol) was dissolved in 40 mL ofa 2 N solution of methyl amine in dry methanol and the reaction mixturewas stirred overnight at room temperature. After this time the solventwas removed under reduced pressure and the residue was purified by flashchromatography (silica gel, 5:1:0.1 chloroform/methanol/concentratedammonium hydroxide) to give free amine 115 (0.25 g, 35%) as a clear oil.¹H NMR (300 MHz, DMSO-d₆) δ 1.58 (m, 4H), 2.55 (m, 2H), 2.73 (m, 2H),3.83 (m, 2H), 3.97 (m, 2H), 6.83 (d, 2H), 7.07 (d, 2H), 7.82 (s, 1H).

4-[4-(2-hydroxyethyloxy)phenyl)]butylamidino-3,5-diamino-6-chloropyrazinecarboxamidehydrochloride (116)

1-(3,5-Diamino-6-chloropyrazinoyl-2-methyl-pseudothiourea hydroiodide(0.32 g, 0.8 mmol) was added to a solution of amine 115 (0.25 g, 1.2mmol) in a mixture of THF (15 mL), methanol (5 mL) anddiisopropylethylamine (1 mL). The reaction mixture was stirred at refluxfor 3.5 h and then cooled to room temperature. The formed precipitatewas isolated, washed with ethyl acetate (2×5 mL) and treated with 5% HCl(10 mL). The resulting solid was isolated by filtration, washed withwater and dried under vacuum to give compound 116 (0.25 g, 73%) as ayellow solid. ¹H NMR (300 MHz, DMSO-d₆) δ 1.56 (m, 4H), 2.57 (m, 2H),3.32 (m, 2H), 3.70 (m, 2H), 3.93 (m, 2H), 4.90 (t, 1H), 6.84 (d, 2H),7.12 (d, 2H), 7.45 (s, 2H), 8.70 (br s, 1H), 8.88 (br s, 1H), 9.12 (brs, 1H) 10.45 (s, 1H). APCI MS m/z=422 [C₁₈H₂₄ClN₇O₃+H]⁺.

Example 184-[4-(2-Hydroxypropyloxy)phenyl)]butylamidino-3,5-diamino-6-chloropyrazinecarboxamidehydrochloride

Using general procedure Z, 4-[4-(2-hydroxypropyloxy)phenyl]butyl aminewas converted into4-[4-(2-hydroxypropyloxy)phenyl)]butylamidino-3,5-diamino-6-chloropyrazinecarboxamidehydrochloride, m.p. 212-214° C., APCI MS, M/Z=436 [C₁₉H₂₆ClN₇O₃+H]⁺.

Example 194-[4-(3-Hydroxypropyloxy)phenyl)]butylamidino-3,5-diamino-6-chloropyrazinecarboxamidehydrochloride

Using general procedure Z, 4-[4-(3-hydroxypropyloxy)phenyl]butyl aminewas converted into4-[4-(3-hydroxypropyloxy)phenyl)]butylamidino-3,5-diamino-6-chloropyrazinecarboxamidehydrochloride, m.p. 211-213° C., APCI MS, M/Z=436 [C₁₉H₂₆ClN₇O₃+H]⁺.

Example 204-[4-(2-{Tetrahydropyan-2-yl}oxyethyloxy)phenyl)]butylamidino-3,5-diamino-6-chloropyrazinecarboxamide

Using general procedure Z,4-[4-(2-{tetrahydropyan-2-yl}oxyethyloxy)phenyl]butyl amine wasconverted into4-[4-(2-{tetrahydropyan-2-yl}oxyethyloxy)phenyl)]butylamidino-3,5-diamino-6-chloropyrazinecarboxamide,m.p. 161° C., APCI Mass Spectrum, M/Z=506 [C₂₃H₃₂ClN₇O₄+H].⁺

Example 214-[3-(2-,3-Dihydroxypropyloxyl)phenyl)]butylamidino-3,5-diamino-6-chloropyrazinecarboxamidehydrochloride

Using general procedure Z,4-[3-(2,3-dihydroxypropyloxy)phenyl]butylamine was converted into4-[3-(2,3-dihydroxypropyloxy)phenyl]butylamidino-3,5-diamino-6-chloropyrazinecarboxamidehydrochloride, m.p. 91-93° C., APCI Mass Spectrum, M/Z=452[C₁₉H₂₆ClN₇O₄+H]⁺.

Example 224-[2-(2-,3-Dihydroxypropyloxy)phenyl)]butylamidino-3,5-diamino-6-chloropyrazinecarboxamidehydrochloride

Using general procedure Z,4-[2-(2,3-dihydroxypropyloxy)phenyl]butylamine was converted into4-[2-(2,3-dihydroxypropyloxy)phenyl]butylamidino-3,5-diamino-6-chloropyrazinecarboxamidehydrochloride, m.p. 200-205° C., APCI Mass Spectrum, M/Z=452[C₁₉H₂₆ClN₇O₄+H]⁺.

Example 234-[4-(2,3,4-Trihydroxybutyloxy)phenyl)]butylamidino-3,5-diamino-6-chloropyrazinecarboxamidehydrochloride

Using general procedure Z,4-[4(2,3,4-trihydroxybutyloxy)phenyl]butylamine was converted into4-[4-(2,3,4-trihydroxybutyloxy)phenyl]butylamidino-3,5-diamino-6-chloropyrazinecarboxamidehydrochloride. m.p. 148° C. (dec), APCI Mass Spectrum, M/Z=482[C₂₀H₂₈ClN₇O₅+H].⁺

Example 244-[4-(4-Amino)phenyl)]butylamidino-3,5-diamino-6-chloropyrazinecarboxamidehydrochloride

Using general procedure Z, 4-[(4-amino)phenyl]butylamine was convertedinto4-[4-(4-amino)phenyl]butylamidino-3,5-diamino-6-chloropyrazinecarboxamidehydrochloride. m.p. 195-200° (dec), APCI Mass Spectrum, M/Z=377[C₁₆H₂₁ClN₈O₄+H⁺].⁺

Example 254-[4-(2-aminoethyloxy)phenyl)butylamidino-3,5-diamino-6-chloropyrazinecarboxamidehydrochloride

Using general procedure Z, 4-[4-(2{t-butoxycarbonylamino}ethyloxy)phenyl]butyl amine was converted into4-[4-(2-{t-butoxycarbonylamino}ethyloxy)phenyl)]butylamidino-3,5-diamino-6-chloropyrazinecarboxamide,m.p. 118° C., APCI MS M/Z=521[C₂₃H₃₃ClN₈O₄+H]⁺, which was hydrolyzed andacidified with HCL to give4-[4-(2-aminoethyloxy)phenyl)butylamidino-3,5-diamino-6-chloropyrazinecarboxamidehydrochloride, m.p.>178° C. (dec), M/Z=421 [C₁₈H₂₅ClN₈O₂].

Example 264-{4-(2-Hydroxyethyl)phenyl)butylamidino-3,5-diamino-6-chloropyrazinecarboxamidehydrochloride

Using general procedure Z, 4-[4-(2-hydroxyethyl)phenyl)]butyl amine wasconverted into4-[4-(2-hydroxyethyl)phenyl)]butylamidino-3,5-diamino-6-chloropyrazinecarboxamidehydrochloride, m.p. 218-219° C., API M/Z=406[C₁₈H₂₄ClN₇O₂H]⁺.

Example 274-[3-(2-Hydroxyethyloxy)phenyl)butylamidino-3,5-diamino-6-chloropyrazinecarboxamidehydrochloride

Using general procedure Z, 4-[3-(2-hydroxyethyloxy)phenyl)butyl aminewas converted to4-[(3-(2-hydroxyethyloxy)phenyl)]butylamidino-3,5-diamino-6-chloropyrazinecarboxamidehydrochloride, m.p. 161-163° C. (dec), AMPI MS M/Z=422[C₁₈H₂₄ClN₇O₃+H].⁺

REFERENCES

-   1. Taylor, E. C.; Harrington, P. M.; Schin, C. Heterocycles, 1989,    28, 1169, incorporated herein by reference-   2. Widsheis et al, Synthesis, 1994, 87-92, incorporated herein by    reference.

Sodium Channel Blocking Activity

The compounds shown in the Tables below were tested for potency incanine bronchial epithelia using the in vitro assay described above.Amiloride was also tested in this assay as a positive control. Theresults for the compounds of the present invention are reported asfold-enhancement values relative to amiloride.

Example 28

Position Fold Enhancement n of R R Over Amiloride 2 4 NH₂ 12.7

Example 29

Fold Enhancement R Over Amiloride H 124

36 H₃CxCH₃* 91 *= the R groups are bonded together via —C(CH₃)₂—

Example 30

Fold Enhancement n Over Amiloride 1 87 2 42 4 28.7

Example 31

Fold Enhancement R⁵ Over Amiloride 40 65 —O—SO₃H 27.7 —O-Glucuronide11.2 Na⁺ Salt —CH₂OH 57 —CO₂CH₃ 26.5

Example 32 Effect of Compound (33) from Example 3 on MCC

These experiments were conducted with compound (33) from Example 3, andthe vehicle as a control. The results are shown in FIGS. 1 (t=0 hours)and 2 (t=4 hours).

Methods Animal Preparation:

Adult ewes (ranging in weight from 25 to 35 kg) were restrained in anupright position in a specialized body harness adapted to a modifiedshopping cart. The animals' heads were immobilized and local anesthesiaof the nasal passage was induced with 2% lidocaine. The animals werethen nasally intubated with a 7.5 mm internal diameter endotracheal tube(ETT). The cuff of the ETT was placed just below the vocal cords and itsposition was verified with a flexible bronchoscope. After intubation theanimals were allowed to equilibrate for approximately 20 minutes priorto initiating measurements of mucociliary clearance.

Administration of Radio-Aerosol:

Aerosols of ^(99m)Tc-Human serum albumin (3.1 mg/ml; containingapproximately 20 mCi) were generated using a Raindrop Nebulizer whichproduces a droplet with a median aerodynamic diameter of 3.6 μm. Thenebulizer was connected to a dosimetry system consisting of a solenoidvalve and a source of compressed air (20 psi). The output of thenebulizer was directed into a plastic T connector; one end of which wasconnected to the endotracheal tube, the other was connected to a pistonrespirator. The system was activated for one second at the onset of therespirator's inspiratory cycle. The respirator was set at a tidal volumeof 500 mL, an inspiratory to expiratory ratio of 1:1, and at a rate of20 breaths per minute to maximize the central airway deposition. Thesheep breathed the radio-labeled aerosol for 5 minutes. A gamma camerawas used to measure the clearance of ^(99m)Tc-Human serum albumin fromthe airways. The camera was positioned above the animal's back with thesheep in a natural upright position supported in a cart so that thefield of image was perpendicular to the animal's spinal cord. Externalradio-labeled markers were placed on the sheep to ensure properalignment under the gamma camera. All images were stored in a computerintegrated with the gamma camera. A region of interest was traced overthe image corresponding to the right lung of the sheep and the countswere recorded. The counts were corrected for decay and expressed aspercentage of radioactivity present in the initial baseline image. Theleft lung was excluded from the analysis because its outlines aresuperimposed over the stomach and counts can be swallowed and enter thestomach as radio-labeled mucus.

Treatment Protocol (Assessment of Activity at t-Zero):

A baseline deposition image was obtained immediately after radio-aerosoladministration. At time zero, after acquisition of the baseline image,vehicle control (distilled water), positive control (amiloride), orexperimental compounds were aerosolized from a 4 ml volume using a PariLC JetPlus nebulizer to free-breathing animals. The nebulizer was drivenby compressed air with a flow of 8 liters per minute. The time todeliver the solution was 10 to 12 minutes. Animals were extubatedimmediately following delivery of the total dose in order to preventfalse elevations in counts caused by aspiration of excess radio-tracerfrom the ETT. Serial images of the lung were obtained at 15-minuteintervals during the first 2 hours after dosing and hourly for the next6 hours after dosing for a total observation period of 8 hours. Awashout period of at least 7 days separated dosing sessions withdifferent experimental agents.

Treatment Protocol (Assessment of Activity at t−4 Hours):

The following variation of the standard protocol was used to assess thedurability of response following a single exposure to vehicle control(distilled water), positive control compounds (amiloride or benzamil),or investigational agents. At time zero, vehicle control (distilledwater), positive control (amiloride), or investigational compounds wereaerosolized from a 4 ml volume using a Pari LC JetPlus nebulizer tofree-breathing animals. The nebulizer was driven by compressed air witha flow of 8 liters per minute. The time to deliver the solution was 10to 12 minutes. Animals were restrained in an upright position in aspecialized body harness for 4 hours. At the end of the 4-hour periodanimals received a single dose of aerosolized ^(99m)Tc-Human serumalbumin (3.1 mg/ml; containing approximately 20 mCi) from a RaindropNebulizer. Animals were extubated immediately following delivery of thetotal dose of radio-tracer. A baseline deposition image was obtainedimmediately after radio-aerosol administration. Serial images of thelung were obtained at 15-minute intervals during the first 2 hours afteradministration of the radio-tracer (representing hours 4 through 6 afterdrug administration) and hourly for the next 2 hours after dosing for atotal observation period of 4 hours. A washout period of at least 7 daysseparated dosing sessions with different experimental agents.

Statistics:

Data were analyzed using SYSTAT for Windows, version 5. Data wereanalyzed using a two-way repeated ANOVA (to assess overall effects),followed by a paried t-test to identify differences between specificpairs. Significance was accepted when P was less than or equal to 0.05.Slope values (calculated from data collected during the initial 45minutes after dosing in the t-zero assessment) for mean MCC curves werecalculated using linear least square regression to assess differences inthe initial rates during the rapid clearance phase.

Example 33 Synthesis ofN-(3,5-diamino-6-chloropyrazine-2-carbonyl)-N′-{4-[4-(2-guanidinoethoxy)-phenyl]butyl}guanidinedihydrochloride (9518)

{4-[4-(2-tert-Butoxycarbonylaminoethoxy)phenyl]butyl}carbamic acidbenzyl ester (1)

Diisopropylazodicarboxylate (132 mL) was added dropwise over 45 minutesto a stirring mixture of [4-(4-hydroxyphenyl)butyl]carbamic acid benzylester (50 g, 0.167 mol), (2-hydroxyethyl)carbamic acid tert-butyl ester(103.4 mL, 0.668 mol), and THF (150 mL) with ice-methanol bath coolingfrom 15 to 35° C. When the exothermic reaction ceased, the cooling bathwas removed, and the reaction was allowed to stir at room temperatureovernight. The solvent was evaporated at reduced pressure, and theresidue was applied to a 1 kg column of silica gel and eluted withmethylene chloride. The chromatography was repeated twice. The residueafter evaporation was washed with hexanes (1.5 L), and the resultingsolid was re-crystallized from a mixture of hexanes and methylenechloride (4:1, 1 L) to afford 54 g (73%) of the pure product as a whitesolid. A second crop yielded an additional 10 g (13%) of the product. ¹HNMR (300 MHz, CDCl₃) δ 1.45 (s, 9H), 1.56 (m, 4H), 2.56 (t, 2H), 3.20(m, 2H), 3.50 (m, 2H), 3.98 (t, 2H), 4.75 (br s, 1H), 5.00 (br s, 1H),5.09 (s, 2H), 6.80 (d, 2H), 7.06 (d, 2H), 7.34 (m, 5H).

{2-[4-(4-Aminobutyl)phenoxy]ethyl}carbamic acid tert-butyl ester (2)

{4-[4-(2-tert-Butoxycarbonylaminoethoxy)phenyl]butyl}carbamic acidbenzyl ester 1 (2.5 g, 5.60 mmol), ethanol (20 mL), and 10% palladium oncarbon (1 g), were subject to one atmosphere of hydrogen for 4 h, andthen allowed to stand overnight. After stirring under nitrogen purge,the catalyst was removed by filtering the reaction mixture throughCelite and rinsing with methylene chloride. Evaporation followed by 2 hunder high vacuum afforded the product (1.7 g, 98%) as an oil. ¹H NMR(300 MHz, CDCl₃) δ 1.45 (s, 9H), 1.47 (m, 2H), 1.61 (m, 2H), 1.75 (br s,2H), 2.56 (t, 2H), 2.71 (t, 2H), 3.51 (m, 2H), 3.99 (t, 2H), 5.07 (br s,1H), 6.80 (d, 2H), 7.08 (d, 2H).

[2-(4-{4-[N′-(3,5-Diamino-6-chloropyrazine-2-carbonyl)guanidino]butyl}phenoxy)ethyl]-carbamicacid tert-butyl ester (3)

{2-[4-(4-Aminobutyl)phenoxy]ethyl}carbamic acid tert-butyl ester 2 (1.7g, 5.5 mmol),1-(3,5-diamino-6-chloropyrazine-2-carbonyl)-2-methylisothioureahydriodide (2.6 g, 6.6 mmol), and triethylamine (3.1 mL) were combinedin THF (18 mL). The reaction was stirred at reflux under argon for 1.5h. The product mixture was allowed to cool, and the solvent wasevaporated. Chromatography (silica gel, methylenechloride/methanol/concentrated ammonium hydroxide, 100:10:1) afforded2.65 g (92%) of the pure product as a yellow foamy solid. ¹H NMR (300MHz, CDCl₃) δ 1.44 (s, 9H), 1.62 (m, 2H), 1.61 (m, 2H), 1.75 (br s, 2H),2.56 (t, 2H), 2.71 (t, 2H), 3.51 (m, 2H), 3.99 (t, 2H), 5.07 (br s, 1H),6.80 (d, 2H), 7.08 (d, 2H).

N-{4-[4-(2-Aminoethoxy)phenyl]butyl}-N′-(3,5-diamino-6-chloropyrazine-2-carbonyl)-guanidine(4, 9308)

[2-(4-{4-[N′-(3,5-Diamino-6-chloropyrazine-2-carbonyl)guanidino]butyl}phenoxy)ethyl]-carbamicacid tert-butyl ester 3 (2.63 g, 5.0 mmol) was dissolved in methanol (25mL). 12N HCl (30 mL) was added in 10 ml portions. After stirring for 1h, the reaction was complete by TLC (silica gel, methylenechloride/methanol/concentrated ammonium hydroxide, 100:10:1). Thesolvent was evaporated and methanol (300 mL) was added and, this processwas repeated. The residue was placed under high vacuum overnight toafford the product (2.65 g, 99%) as a yellow solid, which was usedwithout further manipulation. ¹H NMR (300 MHz, DMSO-d₆) δ 1.58 (m, 4H),2.57 (m, 2H), 3.16 (m, 2H), 3.37 (m, 2H), 4.18 (t, 2H), 6.91 (d, 2H),7.15 (d, 2H), 7.45 (m, 4H), 8.45 (br s, 3H), 9.03 (br s, 2H), 9.46 (t,1H), 10.63 (s, 1H).

N-(4-{4-[2-(N′,N″-Di-tert-butoxycarbouyiguanidino)ethoxy]phenyl}butyl)-N′-(3,5-diamino-6-chloro-pyrazine-2-carbonyl)guanidine(5)

Triethylamine (1.4 mL, 10 mmole) was dripped, via a syringe, into astirring solution ofN-{4-[4-(2-aminoethoxy)phenyl]butyl}-N′-(3,5-diamino-6-chloro-pyrazine-2-carbonyl)guanidine4 (500 mg, 0.943 mmole) in methanol (3 mL). To this stirring solutionwas added 1,3-diBoc-2-(trifluoromethanesulfonyl)guanidine (Goodman'sReagent) (406 mg, 1.04 mmole), and this was allowed to stir at roomtemperature. After 2 h, TLC indicated the absence of Goodman's reagent.An additional 40 mg of the Goodman's reagent was added, and the reactionwas stirred for additional 1 h. After evaporating at below 35° C., thecrude product was chromatographed (silica gel, methylenechloride/methanol/concentrated ammonium hydroxide, 100:10:1) to obtainthe pure product as a yellow solid. ¹H NMR (300 MHz, CDCl₃) δ 1.49 (s,9H), 1.51 (s, 9H), 1.67 (m, 4H), 2.58 (m, 2H), 3.22 (m, 2H), 3.82 (q,2H), 4.05 (t, 2H), 5.22 (br s, 1H), 6.84 (d, 2H), 7.06 (d, 2H), 8.73 (t,1H), 11.47 (br s, 1H).

N-(3,5-Diamino-6-chloropyrazine-2-carbonyl)-N′-{4-[4-(2-guanidinoethoxy)phenyl]butyl}-guanidinedihydrochloride (6)

12N HCl (15 mL) was added dropwise to an ice cooled solution ofN-(4-{4-[2-(N′,N″-di-tert-butoxycarbonylguanidino)ethoxy]phenyl}butyl)-N′-(3,5-diamino-6-chloropyrazine-2-carbonyl)-guanidine5 (370 mg, 0.56 mmole) in methanol (15 mL) over 1 min. After theaddition, the cooling bath was removed, and the reaction was allowed tostir for 30 min. TLC (silica gel, methylenechloride/methanol/concentrated ammonium hydroxide, 3:3:1) indicated slowreaction progression. An additional 15 mL of 12N HCl was added dropwiseat room temperature, and after 2 h, TLC indicated reaction completion.The solvent was evaporated and methanol (100 mL) was added and thenevaporated below 30° C., this process was repeated a further three timesand then the residue was placed under vacuum at 40° C. for 2 days toafford 300 mg (96%) of the pure product as a yellow solid. ¹H NMR (300MHz, DMSO-d₆) δ 1.57 (m, 4H), 2.56 (m, 2H), 3.52 (m, 2H), 4.02 (t, 2H),4.60 (br s, 2H), 6.88 (d, 2H), 7.14 (d, 2H), 7.41 (m, 8H), 7.91 (t, 1H),8.93 (m, 2H), 9.33 (t, 1H), 10.55 (s, 1H). mp 223-230. m/z (ESI)=463[C₁₉H₂₇ClN₁₀O₂+H]⁺.

Example 34N-[4-(4-{2-[bis-((2S,3R)-2,3,4-trihydroxybutyl)amino]ethoxy}phenyl)butyl]-N′-(3,5-diamino-6-chloropyrazine-2-carbonyl)guanidinedihydrochloride (10833)

Method A: via the reaction of {4-[4-(2-aminoethoxy)phenyl]butyl}carbamicacid benzyl ester 7 with D-(−)-erythrose.

{4-[4-(2-Aminoethoxy)phenyl]butyl}carbamic acid benzyl esterhydrochloride (7)

{4-[4-(2-tert-Butoxycarbonylaminoethoxy)phenyl]butyl}carbamic acidbenzyl ester 1 (11.0 g, 24.9 mmol) was dissolved in methanol (110 mL)and THF (20 mL). 12N Hydrochloric acid (40 mL) was added dropwise, andthe reaction was allowed to stir. After 1.5 h, ether (200 mL) was added,and the reaction was suction filtered to collect a white solid. Thesolid was washed with ether, air dried, and dried under vacuum. Thisafforded 7.6 g, (82%) of 7 as a white solid. ¹H NMR (300 MHz, CD₃OD) δ1.39 (br s, 11H), 1.52 (m, 2H),

4-[N,N-bis-((2S,3R)-2,3,4-trihydroxybutyl)-2-aminoethoxy]phenylbutylamine(9)

Acetic acid (0.18 mL, 3.08 mmol) and D-(−)-erythrose (0.74 g, 6.16 mmol)were sequentially added into a suspension of{4-[4-(2-aminoethoxy)phenyl]-butyl}carbamic acid benzyl esterhydrochloride 7 (0.58 g, 1.54 mmol) in methanol (20 mL). The reactionmixture was stirred for 20 minutes at room temperature and undernitrogen atmosphere; then sodium cyanoborohydride (0.39 g, 6.16 mmol)was added at −78° C. The reaction was allowed to warm up to roomtemperature. After overnight stirring, the solvent was evaporated andthe residue was purified by Flash™ (BIOTAGE, Inc) (90 g silica gelcartridge 40M, 5:1:0.1 chloroform/methanol/concentrated ammoniumhydroxide) to provide 8 (0.61 g, 72%) as a white solid. m/z (APCI)=551[C₂₈H₄₂N₂O₉+H]⁺.

The compound 8 (0.30 g, 0.55 mmol) was dissolved in methanol (30 mL) andstirred with 10% palladium on carbon (0.24 g. wet) for 4 h at roomtemperature and atmospheric pressure of hydrogen. The mixture was thenfiltered through a silica gel pad; the solvent was evaporated to provide9 (0.21 g, 92%) as a white solid. ¹H NMR (300 MHz, CD₃OD) δ 1.55 (m,2H), 1.66 (m, 2H,) 2.58 (m, 2H), 2.70 (m, 4H), 2.92 (m, 2H), 2.96 (m,2H), 3.05 (m, 2H), 3.42 (m, 2H), 3.55 (m, 2H), 3.62 (m, 2H), 3.70 (m,2H), 4.10 (m, 2H), 6.86 (d, 2H), 7.08 (d, 2H).

N-[4-(4-{2-[bis-((2S,3R)-2,3,4-trihydroxybutyl)amino]ethoxy}phenyl)butyl]-N′-(3,5-diamino-6-chloropyrazine-2-carbonyl)guanidinedihydrochloride (10)

1-(3,5-Diamino-6-chloropyrazinoyl)-2-methylisothiourea hydriodide (0.196g, 0.5 mmol) and triethylamine (0.077 mL, 0.55 mmol) were sequentiallyadded into a suspension of 9 (0.21 g, 0.5 mmol) in 10 mL of ethanol. Thereaction mixture was stirred at 65° C. for 3 h; the solvent was thenevaporated. The free base of the target compound 10 (0.166 g, 52%) waspurified by Flash™ (BIOTAGE, Inc) (90 g silica gel cartridge 40M,3:1:0.3 chloroform/ethanol/concentrated ammonium hydroxide) as a yellowsolid. It was then treated with 3% HCl (2 mL). The precipitate wascollected by filtration, washed with methylene chloride (4×5 mL), thentaken into water (2 mL) and freeze-dried overnight to provide 152 mg(44%) of 10 as a yellow powder. ¹H NMR (300 MHz, CD₃OD) δ 1.70 (br.s,4H), 2.64 (m, 2H), 3.28 (m, 2H), 3.32 (br.s, 2H), 3.49 (m, 2H),3.55-3.80 (m, 8H), 3.87 (m, 2H), 4.05 (m, 2H), 4.35 (m, 2H), 6.95 (d,2H), 7.15 (d, 2H). m/z (APCI)=629 [C₂₆H₄₁ClN₈O₈+H]⁺, [α]_(D) ²⁵=−12.6°(c=1.03, MeOH).

Method B: via the reaction ofN-{4-[4-(2-aminoethoxy)phenyl]butyl}-N′-(3,5-diamino-6-chloropyrazine-2-carbonyl)guanidine4 with D-(−)-erythrose.

The compound 4 (0.2 g, 0.48 mmol) was suspended in 7 mL of methanol andD-(−)-erythrose (0.17 g, 1.4 mmol) dissolved in 0.9 mL of methanol wasadded. The reaction mixture was stirred at room temperature for 30 min.After this time 25 μL of acetic acid was added to give a clear solution.The reaction mixture was cooled to −78° C. and sodium cyanoborohydride(0.084 g, 1.4 mmol) was added. The reaction was stirred at −78° C. for 2h and at room temperature for 3 d. After this time the solvent wasremoved under reduced pressure and 15 mL of water was added to the oilyresidue. The oil transformed into a yellow solid after 18 h inrefrigerator. The solid was isolated by centrifugation, washed withwater and dissolved in MeOH containing 0.05 mL of TFA. Silica gel(approx. 20 mL) was added and the solvent was removed under reducedpressure. The impregnated silica gel was submitted for purificationusing Flash™ (Biotage Inc., 90 g silica gel cartridge, eluent:chloroform/methanol/ammonium hydroxide=4:1:0.2). The resulting yellowsolid was dissolved in 10 mL of 5% HCl and the solvent was removed underreduced pressure to give compound 10 (100 mg, 30%) as a yellow solid. ¹HNMR (300 MHz, CD₃OD) δ 1.70 (br s, 4H), 2.64 (m, 2H), 3.29-3.70 (m,20H), 4.07 (m, 2H) 4.37 (br s, 2H), 6.95 (d, 2H), 7.15 (d, 2H). m/z(APCI)=629 [C₂₆H₄₁ClN₈O₈+H]⁺.

Methods C: via 2,4-ethylidene-D-erythroseMethod C.1—precursor (11) constructed directly from pre-assembled amine(4)

N-[4-(4-{2-[Bis-((2R,4S,5R)-5-hydroxy-2-methyl[1,3]dioxan-4-ylmethyl)amino]-ethoxy}phenyl)butyl]-N′-(3,5-diamino-6-chloropyrazine-2-carbonyl)-guanidine(11)

The free base 4 (0.15 g, 0.35 mmol) was suspended in 6 mL of methanol.2,4-Ethylidene-D-erythrose¹ (0.15 g, 1.05 mmol) in 2 mL of methanol wasadded, followed by the addition of 20 μL (0.35 mmol) of acetic acid. Themixture was stirred at room temperature until a clear solution wasformed (approx. 10 min). The reaction solution was cooled to −78° C. andsodium cyanoborohydride (0.07 g, 1.05 mmol) was added. The reactionmixture was stirred at −78° C. for 2 h and at room temperature for 2 d.After this time, the solvent was removed under reduced pressure and theresidue purified by Flash™ (Biotage Inc., 90 g silica gel cartridgeeluent: chloroform/methanol/ammonium hydroxide=10:1:0.1) to give 0.17 g(70%) of 11 as a yellow solid. ¹H NMR (300 MHz, CD₃OD) δ 1.21 (m, 6H),1.65 (br s., 4H), 2.59 (m, 2H), 2.84 (m, 2H), 2.92-3.60 (m, 12H), 4.03(m, 4H), 6.84 (d, 2H), 7.10 (d, 2H). m/z (APCI)=681 [C₃₀H₄₅ClN₈O₈+H]⁺.

Method C.2—precursor (11) constructed after side chain assembly

4-(4-{2-[Bis-((2R,4S,5R)-5-hydroxy-2-methyl[1,3]dioxan-4-ylmethyl)amino]ethoxy}phenyl)-butylcarbamicacid benzyl ester (12)

The compound 7 (0.5 g, 1.32 mmol) was suspended in 8 mL of methanol.2,4-Ethylidene-D-erythrose (0.6 g, 4.1 mmol) in 2 mL of methanol wasadded, followed by the addition of 80 μl (1.32 mmol) of acetic acid. Themixture was stirred at room temperature until a clear solution wasformed (approx. 10 min). The reaction solution was cooled to −78° C. andsodium cyanoborohydride (0.260 g, 1.05 mmol) was added. The reactionmixture was stirred at −78° C. for 2 h and at room temperature for 18 h.After this time, the solvent was removed under reduced pressure and thetarget compound 12 (0.6 g, 80%) was isolated by Flash™ (Biotage Inc., 90g silica gel cartridge, eluent: dichloromethane/methanol/ammoniumhydroxide=17:1:0.1) as a clear oil. ¹H NMR (300 MHz, CD₃OD) δ 1.24 (m.,2H), 1.50 (m, 2H), 2.56 (m, 2H), 2.72 (m, 2H), 3.1 (m, 4H), 3.31-3.48(m, 6H), 4.02 (m, 2H), 4.08 (m, 2H), 4.65 (m, 2H), 5.05 (s, 2H), 6.74(d, 2H), 7.0.5 (d, 2H), 7.23 (m, 5H). m/z (APCI)=603 [C₃₂H₄₆ClN₂O₉+H]⁺.

4-(4-{2-[Bis-((2R,4S,5R)-5-hydroxy-2-methyl[1,3]dioxan-4-ylmethyl)amino]ethoxy}phenyl)-butylamine(13)

The protected amine 12 was stirred at room temperature overnight in 25mL of methanol with Pd/C (186 mg, 10% wet) under hydrogen (1 atm). Afterthis time, the catalyst was filtered off and solvent removed underreduced pressure to give amine 13 (0.49 g, 93%) as a clear oil. Thepurity of 13 was confirmed by TLC on silica gel (eluent:chloroform/methanol/ammonium hydroxide=2.5:1:0.1).

N-[4-(4-{2-[Bis-((2R,4S,5R)-5-hydroxy-2-methyl[1,3]dioxan-4-ylmethyl)amino]ethoxy}-phenyl)butyl]-N′-(3,5-diamino-6-chloropyrazine-2-carbonyl)-guanidine(11)

1-(3,5-Diamino-6-chloropyrazinoyl-2-methylisothiourea hydriodide (0.39g, 1.0 mmol) was added to a solution of 13 (0.49 g, 1.07 mmol) in THF (8mL) containing diisopropylethylamine (0.18 mL, 2 mmol). The reactionmixture was stirred at reflux for 2.5 h and at room temperatureovernight. After this time, the solvent was removed under reducedpressure. The brown residue was washed with ether (2×30 mL) andmethylene chloride (2×10 mL). The residue was dissolved in a minimalvolume of methanol (approx. 2 mL) and poured into water. The precipitatewas collected, washed with water and dried overnight to give crude 11(0.5 g) as a yellow solid. The compound 11 (0.4 g, 54%) was purified byflash chromatography on silica gel (eluent: chloroform/methanol/ammoniumhydroxide=10:1:0.1) as a yellow solid. [α]_(D) ²⁵=−20.4° (c=1.0, MeOH).

N-[4-(4-{2-[bis-((2S,3R)-2,3,4-trihydroxybutyl)amino]ethoxy}phenyl)butyl]-N′-(3,5-diamino-6-chloropyrazine-2-carbonyl)guanidinedihydrochloride (10)

The compound 11 (0.18 g, 0.26 mmol) was dissolved in 15 mL of 10% HCland the reaction mixture was stirred at room temperature for 18 h. Afterthis time the solvent was removed under reduced pressure. The resultingresidue was dried overnight and purified by flash chromatography onsilica gel (eluent: chloroform/methanol/ammonium hydroxide=3:1:0.3) togive 66 mg of a yellow solid. The solid was dissolved in 2.5 mL of 5%HCl and the solvent removed under reduced pressure. The residue wasdissolved in 1.5 mL of methanol and the methanol solution was pouredinto 2-propyl alcohol. The formed precipitate was collected, washed withmethylene chloride and dried in vacuum. 34 mg (18%) of dihydrochloride10 was obtained. ¹H NMR (300 MHz, CD₃OD) δ 1.71 (br s., 4H), 2.64 (m,2H), 3.29-3.70 (m, 20H), 4.07 (m, 2H), 4.36 (br s., 2H), 6.96 (d, 2H),7.16 (d, 2H). m/z (APCI)=629 [C₂₆H₄₁ClN₈O₈+H]⁺; [α]_(D) ²⁵=−12.6°(c=1.0, MeOH).

Method D: via (2R,3S)-3,4-epoxybutan-1,2-diol

4-(4-{2-[Bis-((2S,3R)-2,3,4-trihydroxybutyl)amino]ethoxy}phenyl)butylamine(69)

A solution composed of {4-[4-(2-aminoethoxy)phenyl]butyl}carbamic acidtert-butyl ester 16¹ (0.21 g, 0.681 mmol) and(2R,3S)-3,4-epoxybutan-1,2-diol² (68) (0.177 g, 1.702 mmol) in ethanol(5 mL) was heated at 70° C. overnight. It was then slowly cooled to roomtemperature and treated with concentrated hydrochloric acid (12N, 6 mL)at room temperature for 3 hours. The reaction mixture was concentratedunder vacuum. The residue was taken into ethanol (3 mL) and theresulting solution was concentrated again under vacuum. The procedurewas repeated two more times to ensure no aqueous solvent remained. Theresidue was chromatographed over silica gel, eluting with a mixture ofconcentrated ammonium hydroxide (0-10%), methanol (0-30%), and methylenechloride (100-60%), to afford 0.273 g (89%) of the product 69 as acolorless viscous oil. [α]_(D) ²⁵=−33.1° (c 1.0, MeOH). ¹H NMR (300 MHz,CD₃OD) δ 1.49-1.61 (m, 4H), 2.53-2.73 (m, 5H), 2.90-3.04 (m, 4H),3.52-3.73 (m, 17H), 4.07 (t, J=5.7 Hz, 2H), 6.84 (d, J=8.4 Hz, 2H), 7.07(d, J=8.4 Hz, 2H). m/z (APCI)=417 [C₂₀H₃₆N₂O₇+H]⁺.

N-[4-(4-{2-[Bis-((2S,3R)-2,3,4-trihydroxybutyl)amino]ethoxy}phenyl)butyl]-N′-(3,5-diamino-6-chloropyrazine-2-carbonyl)guanidinedihydrochloride (70, ALB 10833)

Compound 69 (0.112 g, 0.269 mmol) was mixed with ethanol (5 mL). Themixture was heated at 65° C. for 15 min to achieve complete dissolution.To the clear solution were sequentially added triethylamine (23 μL,0.161 mmol) and 1-(3,5-diamino-6-chloropyrazinoyl)-2-methylisothioureahydriodide (0.105 g, 0.269 mmol). The mixture was heated at 65° C. foran additional 2 hours. It was, while still warm, slowly added intomethyl tert-butyl ether (MTBE, 25 mL) cooled by an ice bath. A lightyellow precipitation immediately formed upon the addition of thereaction mixture. The suspension was allowed to warm up to roomtemperature by removing the ice bath, and the stirring was continued forone more hour. The solid was vacuum filtered, washed with MTBE (3×5 mL),and tried under vacuum for 4 hours. The dry material (0.155 g) was thensuspended in ethanol (3 mL), and treated with concentrated HCl (1 mL).Water (3 mL) was added to completely dissolve the solid. The resultingsolution was concentrated to dryness under vacuum. The residue was takeninto methanol (2 mL). The resultant methanolic solution was added into2-propyl alcohol (15 mL) at room temperature. The resulting light yellowsuspension was stirred at room temperature overnight. The solid was thenvacuum filtered, washed with 2-propyl alcohol (3×3 mL), and dried undervacuum. 0.093 g (49%) of the compound 70 was obtained as a light yellowsolid. [α]_(D) ²⁵=−13.9° (c 1.0, MeOH). ¹H NMR (300 MHz, DMSO-d₆) δ1.46-1.68 (m, 4H), 2.50-2.57 (m, 2H), 3.26-3.70 (m, 15H), 3.88-4.98 (m,2H), 4.35-4.72 (m, 2H), 4.98-5.10 (br s, 2H), 5.50-6.70 (br s, 2H), 6.93(d, J=8.3 Hz, 2H), 7.16 (d, J=8.3 Hz, 2H), 7.45 (br s, 2H), 7.84-8.06(br s, 1H), 8.61 (br s, 1H), 8.81 (br s, 1H), 8.94 (br s, 1H), 9.25 (brs, 1H), 10.51 (br s, 1H). m/z (APCI)=629 [C₂₆H₄₁ClN₈O₈+H]⁺.

Example 35N-[4-(4-{2-[bis-((2R,3S)-2,3,4-trihydroxybutyl)amino]ethoxy}phenyl)-butyl]-N′-(3,5-diamino-6-chloropyrazine-2-carbonyl)guanidinedihydrochloride (14143, the Enantiomer of 10833)

(2-Bromoethyl)carbamic acid benzyl ester (14)

Benzyl chloroformate (26 mL, 0.176 mole) was added in one portion to astirring mixture of bromoethylamine hydrobromide (44 g, 0.21 mole),triethylamine (64 mL, 0.46 mole), and methylene chloride (1 L) at below−40° C. After the addition, the cooling bath was removed, and thereaction was allowed to stir for 3 h. The mixture was transferred to a 2L separatory funnel, and sequentially washed with water (2×500 mL), 2NHCl (250 mL), and water (500 mL). The resulting solution was suctionfiltered through a 100 g pad of silica gel and washed with methylenechloride. Evaporation of the solvents afforded the product 14 (40 g,89%) as an oil. ¹H NMR (300 MHz, CDCl₃) δ 3.46 (t, 2H), 3.60 (q, 2H),5.11 (s, 2H), 5.20 (br s, 1H), 7.35 (m, 5H).

{4-[4-(2-Benzyloxycarbonylaminoethoxy)phenyl]butyl}carbamic acidtert-butyl ester (15)

[4-(4-Hydroxyphenyl)butyl]carbamic acid tert-butyl ester (38 g, 0.143mole), cesium carbonate (84 g, 0.257 mole), and (2-bromoethyl)carbamicacid benzyl ester 14 (59 g, 0.23 mole) were combined in DMF (200 ml).The mixture was mechanically stirred under nitrogen at 63° C. for 5.5 h,and allowed to stand at room temperature overnight. An additional amountof (2-bromoethyl)carbamic acid benzyl ester 14 (5 g, 0.019 mole) andcesium carbonate (7.1 g, 0.21 mole) were added, and the reaction wasfurther stirred at 65° C. for 1 h. Toluene was then added, and thestirring mixture was allowed to cool to room temperature. The mixturewas suction filtered through a medium sintered glass buchner funnel andwashed with toluene. The solvents were removed under vacuum at 75° C.The remaining oil was washed with hexanes (2×400 mL), then dissolved inether (500 ml) and washed with a mixture of water and brine (10:1, 4×100mL). The remaining solution was suction filtered through a 30 g pad ofsilica gel and the solvent evaporated. The residue was washed withhexanes (3×400 ml), and then placed under vacuum for 2 h to afford 61.8g of the product 15 which was used without further purification. ¹H NMR(300 MHz, CDCl₃) δ 1.45 (s, 9H), 1.54 (m, 4H), 2.56 (t, 2H), 3.20 (m,2H), 3.50 (m, 2H), 3.98 (t, 2H), 4.75 (br s, 1H), 5.00 (br s, 1H), 5.09(s, 2H), 6.80 (d, 2H), 7.06 (d, 2H), 7.34 (m, 5H).

{4-[4-(2-Aminoethoxy)phenyl]butyl}carbamic acid tert-butyl ester (16)

{4-[4-(2-Benzyloxycarbonylaminoethoxy)phenyl]butyl}carbamic acidtert-butyl ester 15 (61.8 g) was stirred in ethanol (500 ml) with 10%palladium on carbon (6 g, wet), under one atmosphere of hydrogen. Afterstirring for more than 6 h, TLC (silica gel, methylene chloride/THF,20:1) indicated reaction completion. The complete reaction was flushedwith nitrogen, and suction filtered through a pad of Celite, and the padwashed with methylene chloride. The residue (41 g) after evaporation ofthe methylene chloride was applied to an 800 g pad of silica gel, andsequentially eluted with a mixture of THF and methylene chloride (1.4 L,2:1), and a mixture of methylene chloride, methanol, and concentratedammonium hydroxide (30:10:1, 2 L). The fraction containing the productwas collected. Evaporation afforded the pure product 16 (32.8 g, 74%over 2 steps). ¹H NMR (300 MHz, CDCl₃) δ 1.44 (s, 9H), 1.50 (br s, 2H),1.55 (m, 4H), 2.56 (t, 2H), 3.12 (m, 2H), 3.50 (m, 2H), 3.98 (t, 2H),4.75 (br s, 1H), 5.00 (br s, 1H), 5.09 (s, 2H), 6.80 (d, 2H), 7.06 (d,2H), 7.34 (m, 5H).

[4-(4-{2-[Bis-((2R,3S)-2,3,4-trihydroxybutyl)amino]ethoxy}phenyl)butyl]carbamicacid tert-butyl ester (18)

A solution composed of the compound 16 (0.4 g, 1.297 mmol) and(2S,3R)-3,4-epoxybutan-1,2-diol² (17) (0.405 g, 3.891 mmol) in ethanol(6 mL) was heated at 60° C. overnight. It was then concentrated undervacuum. The residue was loaded onto silica gel, and eluted by a mixtureof concentrated ammonium hydroxide (0-3%), methanol (0-30%), andmethylene chloride (100-67%) to afford 0.592 g (88%) of the product 18as a colorless viscous oil. [α]_(D) ²⁵=+19.9° (c 0.84, MeOH). ¹H NMR(300 MHz, CD₃OD): δ 1.32-1.67 (m, 13H), 2.54 (t, J=7.1 Hz, 2H),2.72-2.79 (m, 2H), 2.94 (dd, J=13.2 Hz, 3.3 Hz, 2H), 3.02-3.09 (m, 2H),3.51-3.55 (m, 4H), 3.59 (d, J=3.0 Hz, 2H), 3.67-3.74 (m, 4H), 4.08 (t,J=5.6 Hz, 2H), 4.63 (br, 1H), 6.86 (d, J=8.4 Hz, 2H), 7.07 (d, J=8.4 Hz,2H). m/z (APCI)=517 [C₂₅H₄₄N₂O₉+H]⁺.

4-(4-{2-[Bis-((2R,3S)-2,3,4-trihydroxybutyl)amino]ethoxy}phenyl)butylamine(19)

To a solution containing the compound 18 (0.502 g, 0.972 mmol) inethanol (10 mL) was slowly added concentrated hydrochloric acid (12N, 2mL). The clear solution was stirred at room temperature for 4 hours. Thereaction mixture was concentrated under vacuum. The residue was takeninto ethanol (3 mL) and the resulting solution was concentrated againunder vacuum. The procedure was repeated two more times to ensure noaqueous solvent remained. The residue was chromatographed over silicagel, eluting with a mixture of concentrated ammonium hydroxide (0-10%),methane (0-30%), and methylene chloride (100-60%), to afford 0.396 g(98%) of the product 19 as a low melting white solid. [α]_(D) ²⁵=+24.2°(c 0.265, MeOH). ¹H NMR (300 MHz, DMSO-d₆): δ 1.32-1.52 (m, 4H),2.46-2.55 (m, 4H), 2.74-2.79 (m, 2H), 2.84-2.96 (m, 2H), 3.31-3.99 (m,16H), 4.01 (t, J=5.9 Hz, 2H), 6.40 (br s, 2H), 6.82 (d, J=8.5 Hz, 2H),7.09 (d, J=8.5 Hz, 2H). m/z (APCI)=417 [C₂₀H₃₆N₂O₇+H]⁺.

N-[4-(4-{2-[Bis-((2R,3S)-2,3,4-trihydroxybutyl)amino]ethoxy}phenyl)butyl]-N′-(3,5-diamino-6-chloropyrazine-2-carbonyl)guanidine(20, ALB 14143)

The compound 19 (0.15 g, 0.331 mmol) was mixed with ethanol (5 mL). Themixture was heated at 65° C. for 15 min to achieve complete dissolution.To the clear solution were sequentially added di-isopropylethylamine(0.26 mL, 1.505 mmol) and1-(3,5-diamino-6-chloropyrazinoyl)-2-methylisothiourea hydriodide (0.117g, 0.301 mmol). The mixture was heated at 65° C. for an additional 2hours, and subsequently concentrated under vacuum. The residue waschromatographed over silica gel, eluting with a mixture of concentratedammonium hydroxide (1-7%), methanol (10-30%), and methylene chloride(89-63%), to afford 0.152 g (80%) of the free base of 20 as a yellowsolid. mp 78-80° C. (decomposed), [α]_(D) ²⁵=+19.3° (c 1.075, MeOH). ¹HNMR (300 MHz, DMSO-d₆): δ 1.46-1.68 (m, 4H), 2.48-2.62 (m, 2H),2.74-2.95 (m, 4H), 3.08-3.19 (m, 2H), 3.26-3.70 (m, 12H), 4.03 (t, J=5.9Hz, 2H), 4.35-4.72 (m, 6H), 6.60-6.74 (br s, 3H), 6.83 (d, J=8.3 Hz,2H), 7.11 (d, J=8.3 Hz, 2H), 9.05 (br s, 2H). m/z (APCI)=629[C₂₆H_(41Cl)N₈O₈+H]⁺.

A sample of the free base of the compound 20 (0.2 g) was treated with 2NHCl (8 mL). The solution was concentrated to dryness under vacuum. Theresidue was taken into methanol (2 mL). The resultant methanolicsolution was added into 2-propyl alcohol (15 mL) at room temperature,resulting in a light yellow suspension. The solid was vacuum filtered,washed with 2-propyl alcohol (3×3 mL), and dried under vacuum. 0.21 g(94%) of the compound 20 was obtained as a light yellow solid. mp 93-96°C. (decomposed); [α]_(D) ²⁵=+9.06° (c 1.17, MeOH). ¹H NMR (300 MHz,DMSO-d₆): δ 1.46-1.68 (m, 4H), 2.48-2.62 (m, 2H), 3.26-3.70 (m, 17H),4.03 (t, J=5.9 Hz, 2H), 4.35-4.72 (m, 2H), 4.98-5.10 (m 2H), 5.50-6.70(br s, 2H), 6.94 (d, J=8.3 Hz, 2H), 7.14 (d, J=8.3 Hz, 2H), 7.40 (br s,1H), 7.42-7.67 (br s, 1H), 8.68-8.89 (br s, 2H), 9.31 (br s, 1H), 10.53(br s, 1H). m/z (APCI)=629 [C₂₆H₄₁ClN₈O₈+H]⁺.

Example 36N-[4-(4-{2-[(2S,3R)-2,3,4-trihydroxybutylamino]ethoxy}phenyl)butyl]-N-(3,5-diamino-6-chloropyrazine-2-carbonyl)guanidinedihydrochloride (10733)

N-[4-(4-{2-[(2S,3R)-2,3,4-trihydroxybutylamino]ethoxy}phenyl)butyl]-N′-(3,5-diamino-6-chloropyrazine-2-carbonyl)guanidinedihydrochloride (22)

The free base 4 (0.3 g, 0.71 mmol) was suspended in 12 mL of methanoland 0.09 mL (1.4 mmol) of AcOH was added. The mixture was stirred atroom temperature until a clear solution was formed.2,4-Ethylidene-D-erythrose (0.13 g, 0.92 mmol) was then added. Thereaction solution was cooled to −78° C. and sodium cyanoborohydride(0.06 g, 0.92 mmol) was added. The reaction mixture was stirred at −78°C. for 2 h and then at room temperature for 18 h. After this time thesolvent was removed under reduced pressure and the residue purified byFlash™ (Biotage Inc., 90 g silica gel cartridge eluent:chloroform/methanol/ammonium hydroxide=15:1:0.1) to give 0.26 g (66%) of21 as a yellow solid. A sample of the compound 21 (0.2 g, 0.36 mmol) wasthen dissolved in methanol (15 mL) and 300 mg of acidic resin (Dowex 50WX8-200) was added. The mixture was stirred at room temperature for 2 d.After this time, the resin was filtered off and washed with methanol.Then the resin was washed with a 1:1 mixture of MeOH/NH₄OH, (2×20 mL)and filtered off. The supernatants were combined and the solvent wasremoved under reduced pressure. The residue was purified by flashchromatography on silica gel (eluent: chloroform/methanol/ammoniumhydroxide=3:1:0.3). The obtained compound was dried overnight. Afterthis time, the dry residue was dissolved in 5% HCl. Solvent was removedunder reduced pressure and the formed yellow solid was dried overnightto give compound 22 (0.12 g, 55%). ¹H NMR (300 MHz, CD₃OD) δ 1.68 (brs., 4H), 2.64 (m, 2H), 3.15-3.75 (m, 11H), 3.95 (m, 1H), 6.94 (d, 2H),7.18 (d, 2H), 9.23 (m, 1H). m/z (APCI)=525 [C₂₂H₃₃ClN₈O₅+H]⁺.

Example 37N-[4-(4-{2-[bis-((2R,3S,4R)-2,3,4,5-tetrahydroxypentyl)amino]ethoxy}-phenyl)butyl]-N′-(3,5-diamino-6-chloropyrazine-2-carbonyl)guanidinedihydrochloride (4330)

N-[4-(4-{2-[bis-((2R,3S,4R)-2,3,4,5-tetrahydroxypentyl)amino]ethoxy}-phenyl)butyl]-N′-(3,5-diamino-6-chloropyrazine-2-carbonyl)guanidinedihydrochloride (23)

D-(+)-xylose (0.35 g, 2.6 mmol) was added to a solution of hydrochloride4 (0.3 g, 0.65 mmol) in methanol (20 mL) and the mixture was stirred for20 min at room temperature. Then the solution was cooled to −78° C. andsodium cyanoborohydride (0.17 g, 2.6 mmol) was added. The reactionmixture was stirred at −78° C. for 2 h and at room temperature for 4 d.After this time, the solvent was removed under reduced pressure and theresidue was washed with water. The formed yellow solid was isolated anddried under vacuum. Then the residue was re-dissolved in 5% HCl and thesolvent was removed at reduced pressure. The obtained compound wasdissolved in water containing 0.1% TFA and purified by preparative HPLC(C 18 Luna column from Phenomenex 250×21.2 mm, 5μ, isocratic method,water/acetonitrile=80%:20%). The fractions containing the targetcompound were combined and the solvent was removed under reducedpressure. The residue was dissolved in 5% HCl and solvent was removedunder reduced pressure (twice). The resulting yellow powder wasdissolved in water and the solution was lyophylized to give 34 mg (7%)of compound 23 as a yellow solid. ¹H NMR (300 MHz, CD₃OD) δ 1.64 (br s.,4H), 2.62 (m, 2H), 3.30 (m, 4H), 3.35-3.70 (m, 13H), 4.23 (m, 2H), 4.47(m, 2H), 6.95 (d, 2H), 7.15 (d, 2H). m/z (APCI)=689 [C₂₈H₄₅ClN₈O₁₀+H]⁺.[α]_(D) ²⁵=−16.1° (c=0.5, MeOH).

Example 384-{4-[N-(3,5-diamino-6-chloropyrazine-2-carbonyl)guanidino]butyl}-N-(2-hydroxyethyl)benzamidehydrochloride (11180)

The synthesis of 4-(4-Carboxymethylphenyl)butylamine (24) was describedin the previous previously provided experimental details (as compound8).

4-(4-tert-Butoxycarbonylaminobutyl)benzoic acid methyl ester (25)

Di-tert-butyl dicarbonate (1.64 g, 7.51 mmol) was added into thesolution of 24 (1 g, 4.84 mmol) in anhydrous methylene chloride (50 mL).The reaction mixture was stirred overnight under an argon atmosphere atroom temperature. Then the solvent was removed under reduced pressure.The residue was separated by Flash™ (BIOTAGE, Inc) (90 g silica gelcartridge 40M, 3:1 hexane/ethyl acetate) to provide 25 as a white solid(1.35 g, 91%). ¹H NMR (300 MHz, CDCl₃) δ 1.42 (s, 9H), 1.52 (m, 2H),1.64 (m, 2H), 2.69 (m, 2H), 3.13 (m, 2H), 3.90 (s, 3H), 4.57 (br s, 1H),7.22 (d, 2H), 7.95 (d, 2H).

4-(4-tert-butoxycarbonylaminobutyl)benzoic acid (26)

An aqueous (10 mL) solution of sodium hydroxide (0.53 g, 13.18 mmol) wasadded into the solution of 25 (1.35 g, 4.39 mmol) in THF (60 mL) and theresulting solution was stirred at room temperature for 48 h and at 60°C. for 14 h. Then the solvent was removed under reduced pressure. Water(20 mL) was added and pH was adjusted to 7 with HCl. The white solidprecipitate was filtered off, washed with water and dried under vacuum.1.22 g (95%) of white solid 26 was obtained. ¹H NMR (300 MHz, DMSO-d₆) δ1.39 (br s, 11H), 1.52 (m, 2H), 1.64 (m, 2H), 2.92 (m, 2H), 6.84 (m,1H), 7.28 (d, 2H), 7.85 (d, 2H).

{4-[4-(2-Hydroxyethylcarbamoyl)phenyl]butyl}carbamic acid tert-butylester (27)

1,1′-Carbonyldiimidazole (0.6 g, 3.71 mmol) was added into the solutionof 26 (0.91 g, 3.09 mmol) in THF (50 mL). The reaction mixture wasstirred at room temperature overnight under argon atmosphere, thenethanolamine (0.28 mL, 4.64 mmol) was added. The stirring was continuedfor 24 h at room temperature and argon atmosphere. The solvent wasevaporated and the residue was purified by Flash™ (BIOTAGE, Inc) (90 gsilica gel cartridge 40M, 18:1:0.1 chloroform/ethanol/concentratedammonium hydroxide). 0.74 g (71%) of a white solid 27 was isolated. ¹HNMR (300 MHz, CDCl₃) δ 1.40 (s, 9H), 1.48 (m, 2H), 1.60 (m, 2H), 2.62(m, 2H), 3.10 (m, 2H), 3.79 (m, 2H), 4.55 (br s, 1H), 6.74 (m, 1H), 7.18(d, 2H), 7.66 (d, 2H).

4-(4-Aminobutyl)-N-(2-hydroxyethyl)benzamide hydrochloride (28)

A solution of 27 (0.4 g, 1.19 mmol) was stirred at room temperature in amixture of methanol/HCl (1:1, 40 mL). The reaction was finished in 2 haccording to HPLC analysis. The solvent was removed under reducedpressure to provide 0.33 g (98%) 28 as a white solid. ¹H NMR (300 MHz,DMSO-d₆) δ 1.60 (m, 4H), 2.63 (m, 2H), 2.78 (m, 2H), 3.31 (m, 2H), 3.50(m, 2H), 7.28 (d, 2H), 7.80 (d, 2H), 7.97 (br s, 2H), 8.46 (m, 1H).

4-{4-[N-(3,5-Diamino-6-chloropyrazine-2-carbonyl)guanidino]butyl}-N-(2-hydroxy-ethyl)benzamidehydrochloride (29)

1-(3,5-Diamino-6-chloropyrazinoyl)-2-methylisothiourea hydriodide (0.48g, 1.24 mmol) and triethylamine (0.7 mL, 4.74 mmol) were sequentiallyadded into a solution of 28 (0.28 g, 1.19 mmol) in a mixture of THF/MeOH(4 mL, 1/1). The reaction mixture was stirred in the boiling solvent for4 h, then at room temperature overnight. The solvent was evaporated. Thefree base of the target compound 29 (0.36 g, 62%) was purified by Flash™(BIOTAGE, Inc) (90 g silica gel cartridge 40M, 12:1:0.1chloroform/ethanol/concentrated ammonium hydroxide) as a yellow solid.100 mg of the yellow solid was treated with 2 mL of 3% HCl. Theprecipitate was collected by filtration, washed with water (2×5 mL) anddried under vacuum to give 85 mg (79%) of 29 as a yellow powder. ¹H NMR(300 MHz, DMSO-d₆) δ 1.60 (m, 4H), 2.68 (m, 2H), 3.28 (m, 4H), 3.49 (m,2H), 4.80 (m, 1H), 7.28 (d, 2H), 7.44 (br s, 2H), 7.82 (d, 2H), 8.45 (m,1H). m/z (APCI)=449 [C₁₉H₂₅ClN₈O₃+H]⁺.

Example 392-{4-[N′-(3,5-diamino-6-chloropyrazine-2-carbonyl)guanidino]-4-butylphenoxy}acetamidehydrochloride (9714)

[4-(4-Benzyloxycarbonylaminobutyl)phenoxy]acetic acid ethyl ester (47)

Sodium hydride (60% dispersion in mineral oil) (0.24 g, 10.05 mmol) wasadded to a cold (0° C.) solution of 4-(4-hydroxyphenyl)butylamine (2 g,6.68 mmol) in THF (150 mL) under nitrogen atmosphere. The reactionmixture was allowed to warm up to room temperature over 0.5 h withstirring, then ethyl bromoacetate (0.96 mL, 8.02 mmol) andtetrabutylammonium iodide (0.25 g, 0.67 mmol) was sequentially added.The reaction was further stirred at room temperature overnight. Silicagel (25 mL) was added into the mixture and the solvent was evaporated.The impregnated silica gel was subjected to column chromatographypurification (silica gel, 5:1 hexanes/ethyl acetate). 2.42 g (94%) of 47was obtained as a white solid. ¹H NMR (300 MHz, CDCl₃) δ 1.30 (t, 3H),1.57 (m, 4H), 2.58 (m, 2H), 3.20 (m, 2H), 4.28 (m, 2H), 4.58 (s, 2H),4.74 (br s, 1H), 5.10 (br s, 2H), 6.82 (m, 2H), 7.08 (m, 2H), 7.38 (brs, 5H).

[4-(4-Aminobutyl)phenoxy]acetic acid ethyl ester (48)

A suspension of 47 (1.11 g, 2.88 mmol) and 10% palladium on carbon (0.40g, wet) in methanol (50 mL) was stirred at room temperature for 2 hunder atmospheric pressure of hydrogen. The mixture was then filteredthrough a silica gel pad. The solvent was evaporated to provide 48 (0.64g, 88%) as a white solid. ¹H NMR (300 MHz, DMSO-d₆) δ 1.21 (m, 3H),1.30-1.63 (m, 6H), 3.13 (m, 2H), 4.17 (m, 2H), 4.72 (br s, 2H), 6.84 (m,2H), 7.10 (m, 2H).

(4-{4-[N′-(3,5-Diamino-6-chloropyrazine-2-carbonyl)guanidino]butyl}phenoxy)aceticacid ethyl ester (49)

1-(3,5-Diamino-6-chloropyrazinoyl)-2-methylisothiourea hydriodide (0.74g, 1.9 mmol) and triethylamine (0.5 mL) were sequentially added into asolution of 48 (0.62 g, 2.47 mmol) in THF (10 mL). The reaction mixturewas stirred in the boiling solvent for 4 h and at room temperatureovernight. The solvent was evaporated and the residue was purified bycolumn chromatography (silica gel, 6:1:0.1chloroform/ethanol/concentrated ammonium hydroxide) to provide 49 (0.5g, 57%) as a yellow solid. The purity of the product was confirmed byHPLC. m/z (APCI)=464 [C₂₀H₂₆ClN₇O₄+H]⁺.

2-{4-[N′-(3,5-Diamino-6-chloropyrazine-2-carbonyl)guanidino]-4-butylphenoxy}acetamidehydrochloride (50, 9714)

A solution of 49 (0.5 g, 1.08 mmol) in ammonia-saturated ethanol (100mL) was stirred at room temperature overnight. The solvent wasevaporated and the residue was purified by column chromatography (silicagel, 4:1:0.1 chloroform/ethanol/concentrated ammonium hydroxide) toafford the free base of the product 50 as a yellow solid. It was thentreated with 3% HCl. The solvent was evaporated. The resulting solid waswashed with water (2×5 mL), and then dried in vacuum to provide 50 (0.25g, 49%) as a yellow solid. ¹H NMR (300 MHz, DMSO-d₆) δ 1.57 (br s, 4H),2.51 (m, 2H), 3.33 (m, 2H), 4.48 (s, 2H), 6.87 (d, 2H), 7.13 (d, 2H),7.37-7.60 (m, 4H), 8.88 (br s, 1H), 8.99 (br s, 1H), 9.32 (m, 1H), 10.56(s, 1H). m/z (APCI)=435.3 [C₁₈H₂₃ClN₈O₃+H]⁺.

Example 404-{4-[N′-(3,5-Diamino-6-chloropyrazine-2-carbonyl)guanidino]butyl}benzamidine(11157)

[4-(4-Cyanophenyl)but-3-ynyl]carbamic acid tert-butyl ester (56)

But-3-ynylcarbamic acid tert-butyl ester (3.66 g, 22 mmol) was addeddropwise to an ice cooled, stirring, argon purged mixture of4-iodobenzonitrile (4.5 g, 19.6 mmol),dichlorobis(triphenylphosphine)palladium(II) (0.69 g, 0.98 mmol), copper(1) iodide (0.19 g, 0.98 mmol), triethylamine (11 mL, 78.4 mmol), andTHF (24 mL). After stirring for 10 min, the ice bath was removed, andthe reaction was allowed to stir for an additional 2 h. The reactionmixture was passed through a pad of silica gel with methylenechloride/ethyl acetate (5:1) as eluant. After evaporating the solvent,the crude product was chromatographed with methylene chloride/ethylacetate (20:1) as eluant. Evaporation of the solvent, followed byplacement under vacuum for 1 h, afforded the pure product 56 (5.2 g,99%) as an oil. ¹H NMR (300 MHz, CDCl₃) δ 1.46 (s, 9H), 2.64 (t, 2H),3.37 (m, 2H), 4.85 (br s, 1H), 7.47 (d, 2H), 7.58 (d, 2H).

[4-(4-Cyanophenyl)butyl]carbamic acid tert-butyl ester (57)

A suspension of [4-(4-cyanophenyl)but-3-ynyl]carbamic acid tert-butylester 56 (5.2 g, 19.2 mmol) and 10% palladium on carbon (2.5 g, wet) inethanol/THE (30 mL, 1:1) was stirred overnight under 1 atmosphere ofhydrogen. After purging with nitrogen, the reaction mixture was suctionfiltered through a pad of Celite. The solvent was removed from thefiltrate by evaporation. The residue was chromatographed on silica gel,eluting with methylene chloride/ethyl acetate (30:1), to afford the pureproduct 57 (4.6 g, 87%) as an oil. ¹H NMR (300 MHz, CDCl₃) δ 1.44 (s,9H), 1.51 (m, 2H), 1.65 (m, 2H), 2.69 (t, 2H), 3.14 (m, 2H), 4.52 (br s,1H), 7.27 (d, 2H), 7.56 (d, 2H).

[4-(4-Thiocarbamoylphenyl)butyl]carbamic acid tert-butyl ester (58)

Nitrogen was bubbled through a stirring solution of[4-(4-cyanophenyl)butyl]carbamic acid tert-butyl ester (57) (4.5 g, 16.4mmol), pyridine (60 mL), and triethylamine (60 mL) for 10 min. Hydrogensulfide was slowly bubbled through this stirring solution for 10 min.The reaction was sealed, and allowed to stir overnight. The reactionmixture was then purged with nitrogen, transferred to a separatoryfunnel with ethyl acetate (500 mL), and sequentially washed with water(3×100 mL), saturated aqueous solution of potassium hydrogen sulfate(3×100 mL), water (2×50 mL), and brine (2×50 mL). The solution was driedover sodium sulfate. The solid was filtered, and the filtrate wasconcentrated under reduced pressure. The resulting solid wasre-crystallized from hexanes/ethyl acetate (10:1), and placed undervacuum for 2 h to afford the pure product 58 (4.8 g, 95%) as a yellowcrystalline solid. ¹H NMR (300 MHz, CDCl₃) δ 1.43 (s, 9H), 1.49 (m, 2H),1.63 (m, 2H), 2.65 (t, 2H), 3.11 (m, 2H), 4.57 (br s, 1H), 7.19 (d, 2H),7.42 (br s, 1H), 7.81 (d, 2H).

[4-(4-Carbamimidoylphenyl)butyl]carbamic acid tert-butyl ester (59)

[4-(4-Thiocarbamoylphenyl)butyl]carbamic acid tert-butyl ester (58) (500mg, 1.6 mmol) and iodomethane (4 mL, 64 mmol) were combined in methylenechloride (8 mL). The solution was stirred at reflux for 3 h, and allowedto stand overnight. The volatiles were removed by evaporation, and theresidue was dried under vacuum for 3 h. The resulting crystalline solidwas dissolved in ethanol (5 mL), and ammonium acetate (1.1 g, 14.4 mmol)was added. The resulting solution was stirred at reflux for 2 h, and thesolvent was evaporated. The residue was taken up in a mixture ofmethanol and concentrated ammonium hydroxide (20 mL, 10:1), and thesolvent was then evaporated. To this residue were added water (20 mL)and concentrated ammonium hydroxide (3 mL). The resulting mixture wasstirred for 1 h, cooled in an ice bath, and suction filtered to collecta solid. The solid was dried under vacuum overnight to afford theproduct 59 (137 mg, 29%). ¹H NMR (300 MHz, DMSO-d₆) δ 1.37 (s, 9H), 1.38(m, 2H), 1.55 (m, 2H), 2.64 (t, 2H), 2.93 (m, 2H), 6.80 (br s, 1H), 7.35(d, 2H), 7.70 (d, 2H), 9.62 (br s, 3H).

4-(4-Aminobutyl)benzamidine dihydrochloride (60)

12 N hydrochloric acid (0.71 mL, 8.6 mmol) was added dropwise into astirring solution of [4-(4-carbamimidoylphenyl)butyl]carbamic acidtert-butyl ester (59) (124 mg, 0.43 mmol) in methanol (2 mL). Afterstirring for 2.5 h, TLC (methylene chloride/methanol/concentratedammonium hydroxide, 6:3:1) indicated reaction completion. The reactionmixture was vacuum filtered. The solvent was removed from the filtrateby evaporation. Residual water was further removed as an azeotrope oftoluene/methanol (1:1). Placement of the residue under vacuum for 2 hafforded the product 60 (106 mg, 94%) as a yellow foamy solid. ¹H NMR(300 MHz, DMSO-d₆) δ 1.62 (m, 4H), 2.70 (t, 2H), 2.78 (m, 2H), 3.49 (brs, 1H), 7.47 (d, 2H), 7.82 (d, 2H), 8.13 (br s, 3H), 9.25 (s, 2H), 9.42(s, 2H).

4-{4-[N′-(3,5-Diamino-6-chloropyrazine-2-carbonyl)guanidino]butyl}benzamidine(61, ALB 11157)

4-(4-Aminobutyl)benzamidine dihydrochloride (60) (92 mg, 0.35 mmol),triethylamine (0.24 mL, 1.74 mmol),1-(3,5-diamino-6-chloropyrazinoyl)-2-methylisothiourea hydriodide (142mg, 0.37 mmol) were sequentially added into ethanol (2 mL). Afterstirring at reflux for 2 h with argon protection, the solvent wasevaporated. The residue was stirred in methylene chloride (5 mL), andsuction filtered to obtain a solid. The solid was chromatographed onsilica gel, eluting with methylene chloride/methanol/concentratedammonium hydroxide (6:3:1), to afford the pure product 61 as a yellowsolid. mp 140-170° C. (decomposed). ¹H NMR (300 MHz, DMSO-d₆) δ 1.96 (m,2H), 2.09 (m, 2H), 3.43 (m, 2H), 4.08 (br s, 2H), 8.00-10.00 (m, 14H).m/z (APCI)=404 (C₁₇H₂₂ClN₉O+H)⁺.

REFERENCES

-   1. Rappoport, D. A.; Hassid, Z.; J. Amer. Chem. Soc., 1951, 73,    5524-5525, incorporated herein by reference, Ruth, J. A. and    Claffey, D J., Tetrahedron Lett. 1996, 37 (44), 7929-7932,    incorporated herein by reference.

Sodium Channel Blocking Activity

The compounds shown in the Table below were tested for potency in caninebronchial epithelia using the in vitro assay described above. Amiloridewas also tested in this assay as a positive control. The results for thecompounds of the present invention are reported as fold-enhancementvalues relative to amiloride.

Example 41

R = N = Fold Amiloride* OH 1  50.9 ± 19.8 (3) OH 2  79.2 ± 30.6 (4) OH 4 45.3 ± 27.0 (6) NH₂ 0  32.6 ± 2.0 (3) NH₂ 1  26.2 ± 5.1 (3) NH₂ 3   59± 5.5 (4) NH₂ 4 132.6 ± 47.2 (5)

Example 42

R = n = Fold Amiloride* OH 2  84.9 ± 30.3 (6) OH 3 105.2 ± 26.6 (7) OH 421 (1) NH₂ 2  60.1 ± 1.3 (2) NH₂ 2  56.5 ± 0 (4) NH₂ 3 102.6 ± 49 (2)

Example 43

Fold X = Y = Amiloride* C = 0 NH₂ 73.1 ± 31.5 (3) C = 0 NH(CH₂)₂—OH 28.5(1) C═NH NH₂ 53.2 ± 19.3 (2) NH H 32.6 ± 2 (3) NH COCH₃ 52.3 ± 16.4 (3)NH SO₂CH₃ 38.5 ± 4.2 (3) NH CO₂C₂H₅ 29.0 ± 5.8 (2) NH C(═NH)NH₂ 88.0 ±18.0 (2)

Example 44

Fold R = R¹ = Amiloride* OR ¹ H 50.9 ± 19.8 (3) NHR ¹ H 28 (1) NHR ¹COCH₃ 16 (1) NHR ¹ SO₂CH₃ 50.6 ± 11.9 (2) NHR ¹ CO₂C₂H₅ 24.1 ± 0.5 (3)NHR ¹ CO₂—(CH₃)₃ 29.0 ± 4.1 (2) NHR C(═NH)NH₂ 66.2 ± 27.4 (4)

Example 45

X = Fold Amiloride* NH₂  56.5 ± 24 (4) NH—C(═NH)—NH₂ 120.6 ± 60.8 (11)NHSO₂CH₃ 64.0 (1) NHCO₂(CH₃)₃  51.7 ± 10.1 (2) NHCOCH₃  48.5 ± 26.5 (4)

Example 46

R = Fold Amiloride* —OH 14.0 ± 4.6 (7) —O(CH₃)₃ 29.2 ± 10.9 (3) —NH₂48.2 ± 24.1 (7)

Example 47

Fold Amiloride* CH₂ CH₂ ÖH  79.2 ± 30.6 (4) O CH₂ CH₂ OH  84.9 ± 30.3(6) O CH₂ CH₂ CH₂ OH 105.2 ± 26.6 CH₂ CH₂ CH₂ CH₂ OH 37.4 (1) O CH₂ CHOHCH₂ OH 51.95 (50) O CH₂ CHOH CH₂ NH₂  57.5 ± 24.5 (6) O CH₂ CH₂ CH₂ CH₂OH 21 (1) O CH₂ CH₂ CHOH CH₂ OH  55.5 ± 19.3 (3) O CH₂ CHOH CHOH CH₂ OH 93.7 ± 42.1 (5) O CH₂ CHOH CHOH CH₂ OH  56.1 ± 15.6 (4) N CH₂ CHOH CHOHCH₂ OH  44.9 ± 14.7 (8)

Example 48

Fold Amiloride* NH₂ 32.6 ± 2 (3) R 44.9 ± 14.7 NH (8) O CH₂ CH₂ NH₂ 84.9± 30.3 (6) O CH₂ CH₂ NH R^(a) 52.9 ± 14.3 (5) O CH₂ CH₂ NR R^(a, c) 73.2± 49.3 (9) O CH₂ CH₂ O R¹ 76.1 (1) O CH₂ CH₂ O CH₃ 51.5 ± 14.9 (2) O R93.7 ± 42.1 (5) O CH₂ CHOH CH₂ OH 51.95 (79) O CH₂ CH₂ NR R^(a, c) 56.0(1) O CH₂ CH₂ NR² R^(2, a) ^(a)Chiral ^(b) Racemic ^(c) Enantiomers

Example 49

Example 50

Fold Amiloride* O(CH₂)NHCO₂ ⁺ 51.7 ± 10.1 (2) OCH₂CO₂ ⁺ 29.2 ± 10.9 (3)OCH₂CO₂Et 20 (1) —NHCH₂CO₂ ⁺ 29.0 ± 4.1 (2) NHCO₂ET 29.0 ± 5.38 (2)CH₂NHCO₂ET 24.1 ± 0.5 (3) O(CH₂)₂NHCO₂ET 17.7 ± 6.0 (2) OCH₂CHOHCH₂NHCO₂⁺ 77.9 ± 24.0 (3) O(CH₂)₃NHCO₂ ⁺ 37.5 ± 12.8 (4) (CH₂)₄—NHCO₂ ⁺ 16.9 ±2.3 (2)

R = Position Fold Amiloride* H Ortho 21.7 ± 4.8 (2) H Meta 41.1 ± 8.5(2) H Para 80.3 ± 25.5 (9) CH₂OH Ortho 24.0 ± 1.0 (2) CH₂OH Meta 40 (1)CH₂OH Para 51.55 (79)

Example 51

R =/Z = H O(CH₂)₂—R O(CH₂)₃—R CH₂R (CH₂)₃R OH Xamiloride 84.9 ± 105.2 ±50.9 ± 30.3  26.6 19.8 R =/Z = H O(CH₂)₂—R O(CH₂)₃—R CH₂R —(CH₂)₃R NH₂Xamiloride 32.6 ± 56.5 ± 102.6 ± 26.2 ± 54.4 ±  2  0  49  5.1 (3) 43.5(6) R =/Z = H O(CH₂)₂—R O(CH₂)₃—R CH₂R (CH₂)₃R

Xamiloride 88.0 ± 98.0 ± 50.2 ± 35 (1) 47.6 (3) 18.0 58.5(18) 17.4(4)

Example 52 Effect of Compound 9518 on MCC

These experiments were conducted according to methods of Example 32 withcompound 9518 and the vehicle as a control The results are shown inFIGS. 3 (t=0 hours) and 4 (t=4 hours).

Example 53 Effect of Compound 9714 on MCC

These experiments were conducted according to methods of Example 32 withcompound 9714 and the vehicle as a control. The results are shown inFIGS. 5 (t=0 hours) and 6 (t=4 hours).

Example 54 Effect of Compound 10833 on MCC

These experiments were conducted according to methods of Example 32 withcompound 10833 and the vehicle as a control The results are shown inFIGS. 7 (t=0 hours) and 8 (t=4 hours).

Obviously, numerous modifications and variations of the presentinvention are possible in light of the above teachings. It is thereforeto be understood that within the scope of the appended claims, theinvention may be practiced otherwise than as specifically describedherein.

1. A compound represented by formula (I):

wherein X is hydrogen, halogen, trifluoromethyl, lower alkyl,unsubstituted or substituted phenyl, lower alkyl-thio, phenyl-loweralkyl-thio, lower alkyl-sulfonyl, or phenyl-lower alkyl-sulfonyl; Y ishydrogen, hydroxyl, mercapto, lower alkoxy, lower alkyl-thio, halogen,lower alkyl, unsubstituted or substituted mononuclear aryl, or —N(R²)₂;R¹ is hydrogen or lower alkyl; each R² is, independently, —R⁷,—(CH₂)_(m)—OR⁸, —(CH₂)_(m)—NR⁷R¹⁰, —(CH₂)_(n)(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸,—(CH₂CH₂O)_(m)—R⁸, —(CH₂CH₂O)_(m)—CH₂CH₂NR⁷R¹⁰, —(CH₂)_(n)—C(═O)NR⁷R¹⁰,—(CH₂)_(n)-Z_(g)—R⁷, —(CH₂)_(m)—NR¹⁰—CH₂(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸,—(CH₂)_(n)—CO₂R⁷, or

R³ and R⁴ are each, independently, hydrogen, a group represented byformula (A), lower alkyl, hydroxy lower alkyl, phenyl, phenyl-loweralkyl, (halophenyl)-lower alkyl, lower-(alkylphenylalkyl), lower(alkoxyphenyl)-lower alkyl, naphthyl-lower alkyl, or pyridyl-loweralkyl, with the proviso that at least one of R³ and R⁴ is a grouprepresented by formula (A):

wherein each R^(L) is, independently, —R⁷, —(CH₂)_(n)—OR⁸,—O—(CH₂)_(m)—OR⁸, —(CH₂)_(n)—NR⁷R¹⁰, —O—(CH₂)_(m)—NR⁷R¹⁰,—(CH₂)_(n)(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸,—O—(CH₂)_(m)(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸, —(CH₂CH₂O)_(m)—R⁸,—O—(CH₂CH₂O)_(m)—R⁸, —(CH₂CH₂O)_(m)—CH₂CH₂NR⁷R¹⁰,—O—(CH₂CH₂O)_(m)—CH₂CH₂NR⁷R¹⁰, —(CH₂)_(n)—C(═O)NR⁷R¹⁰,—O—(CH₂)_(m)—C(═O)NR⁷R¹⁰, —(CH₂)_(n)-(Z)_(g)—R⁷,—O—(CH₂)_(m)-(Z)_(g)—R⁷, —(CH₂)_(n)—NR¹⁰—CH₂(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸,—O—(CH₂)_(m)—NR¹⁰—CH₂(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸, —(CH₂)_(n)—CO₂R⁷,—O—(CH₂)_(m)—CO₂R⁷, —OSO₃H, —O-glucuronide, —O-glucose,

each o is, independently, an integer from 0 to 10; each p is an integerfrom 0 to 10; with the proviso that the sum of o and p in eachcontiguous chain is from 1 to 10; each x is, independently, O, NR¹⁰,C(═O), CHOH, C(═N—R¹⁰), CHNR⁷R¹⁰, or represents a single bond; each R⁵is, independently, —(CH₂)_(m)—OR⁸, —O—(CH₂)_(m)—OR⁸, —(CH₂)_(n)—NR⁷R¹⁰,—O—(CH₂)_(m)—NR⁷R¹⁰, —(CH₂)_(n)(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸,—O—(CH₂)_(m)(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸, —(CH₂CH₂O)_(m)—R⁸,—O—(CH₂CH₂O)_(m)—R⁸, —(CH₂CH₂O)_(m)—CH₂CH₂NR⁷R¹⁰,—O—(CH₂CH₂O)_(m)—CH₂CH₂NR⁷R¹⁰, —(CH₂)_(n)—C(═O)NR⁷R¹⁰,—O—(CH₂)_(m)—C(═O)NR⁷R¹⁰, —(CH₂)_(n)-(Z)_(g)—R⁷,—O—(CH₂)_(m)-(Z)_(g)—R⁷, —(CH₂)_(n)—NR¹⁰—CH₂(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸,—O—(CH₂)_(m)—NR¹⁰—CH₂(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸, —(CH₂)_(n)—CO₂R⁷,—O—(CH₂)_(m)—CO₂R⁷, —OSO₃H, —O-glucuronide, —O-glucose,

each R⁶ is, independently, —R⁷, —OR¹¹, —N(R⁷)₂, —(CH₂)_(m)—OR⁸,—O—(CH₂)_(m)—OR⁸, —(CH₂)_(n)—NR⁷R¹⁰, —O—(CH₂)_(m)—NR⁷R¹⁰,—(CH₂)_(n)(CHOR⁸)(CHOR⁸)n-CH₂OR⁸, —O—(CH₂)_(m)(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸,—(CH₂CH₂O)_(m)—R⁸, —O—(CH₂CH₂O)_(m)—R⁸, —(CH₂CH₂O)m-CH₂CH₂NR⁷R¹⁰,—O—(CH₂CH₂O)_(m)—CH₂CH₂NR⁷R¹⁰, —(CH₂)_(n)—C(═O)NR⁷R¹⁰,—O—(CH₂)_(m)—C(═O)NR⁷R¹⁰, —(CH₂)n-(Z)_(g)—R⁷, —O—(CH₂)_(m)-(Z)_(g)—R⁷,—(CH₂)_(n)—NR¹⁰—CH₂(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸,—O—(CH₂)_(m)—NR¹⁰—CH₂(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸, —(CH₂)_(n)—CO₂R⁷,—O—(CH₂)_(m)—CO₂R⁷, —OSO₃H, —O-glucuronide, —O-glucose,

wherein when two R⁶ are —OR¹¹ and are located adjacent to each other ona phenyl ring, the alkyl moieties of the two R⁶ may be bonded togetherto form a methylenedioxy group; each R⁷ is, independently, hydrogen orlower alkyl; each R⁸ is, independently, hydrogen, lower alkyl,—C(═O)—R¹¹, glucuronide, 2-tetrahydropyranyl, or

each R⁹ is, independently, —CO₂R⁷, —CON(R⁷)₂, —SO₂CH₃, or —C(═O)R⁷; eachR¹⁰ is, independently, —H, —SO₂CH₃, —CO₂R⁷, —C(═O)NR⁷R⁹, —C(═O)R⁷, or—CH₂—(CHOH)_(n)—CH₂OH; each Z is, independently, CHOH, C(═O), CHNR⁷R¹⁰,C═NR¹⁰, or NR¹⁰; each R¹¹ is, independently, lower alkyl; each g is,independently, an integer from 1 to 6; each m is, independently, aninteger from 1 to 7; each n is, independently, an integer from 0 to 7;each Q is, independently, C—R⁵ or C—R⁶, wherein one Q is C—R⁵; or apharmaceutically acceptable salt thereof, and inclusive of allenantiomers, diastereomers, and racemic mixtures thereof.